CN106715465A - Polypeptides with lipase activity and polynucleotides encoding same - Google Patents

Polypeptides with lipase activity and polynucleotides encoding same Download PDF

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Publication number
CN106715465A
CN106715465A CN201580019430.0A CN201580019430A CN106715465A CN 106715465 A CN106715465 A CN 106715465A CN 201580019430 A CN201580019430 A CN 201580019430A CN 106715465 A CN106715465 A CN 106715465A
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polypeptide
acid
composition
seq
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CN106715465B (en
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李明
孙天齐
蒋先芝
韩步聪
C·H·汉森
K·博尔奇
L·鲍恩斯高
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Novo Nordisk AS
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    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
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Abstract

Provided are isolated polypeptides with lipase activity. Also provided are polynucleotides encoding the polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; compositions comprising the polynucleotides and methods of using the polypeptides or the compositions.

Description

Polypeptide and their polynucleotides of coding with lipase active
To the reference of sequence table
The application includes the sequence table of computer-reader form, is incorporated herein by reference.
Background of invention
Invention field
The present invention relates to the polypeptide with lipase active, encode the polynucleotides of these polypeptides, produce these polypeptides Method and the method using these polypeptides.
Description of Related Art
Lipase is important biocatalyst, its shown can be used for it is various application and substantial amounts of different lipase It has been commercialized through identified and many.However, being suitable for making in the different components of currently used condition are adapted to New fats enzyme is desirable.
Lipase has been used in composition, thus for by hydrolyzing triglyceride producing aliphatic acid to make a return journey grease removal Matter spot.Current cleaning and/or Fabrid care composition include many active components, these compositions interference lipase removal lipid The ability of spot.Accordingly, it would be desirable to the lipase that can be worked in for the harsh environment of the composition for cleaning.The application's Purpose is to provide the lipase with favorable characteristics, the fat such as being had improved properties when applying in cleanser compositions Enzyme.
The SEQ ID NO of known lipase and the application from Rhizopus oryzae (UNIPROT B1Q560):2 maturation is more Peptide has 55% uniformity.
Summary of the invention
The present invention relates to the polypeptide of the separation with lipase active, these separate polypeptides are selected from the group, the group by with Lower every composition：(a) and SEQ ID NO:2 mature polypeptide have at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%th, the polypeptide of at least 97%, at least 98%, at least 99% or 100% sequence identity；B () one kind is by following polynucleotides The polypeptide of coding, the polynucleotides under low stringency condition, under middle stringent condition, in-high stringency conditions under, under high stringency conditions Or very under high stringency conditions with (i) SEQ ID NO:1 or SEQ ID NO:3 or (i) total length complement hybridization；C () is a kind of Polypeptide, the polypeptide by with SEQ ID NO:1 or SEQ ID NO:3 mature polypeptide encoded sequence have at least 60%, at least 65%th, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, The multinuclear of at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity Thuja acid is encoded；A kind of (d) polypeptide, the polypeptide be at one or more (for example, a number) position include substitution, missing and/ Or the SEQ ID NO of insertion:2 variant；And a kind of (e) polypeptide, the polypeptide be (a), (b), (c) or (d) polypeptide in appoint The fragment of one.
Polynucleotides the invention further relates to encode the separation of these polypeptides；Nucleic acid construct comprising these polynucleotides Body, carrier and host cell；And produce the method for the polypeptide and the purposes of the polypeptide.
Method the invention further relates to include the composition of the polypeptide and cleaned using the polypeptide.
Sequence table is summarized
SEQ ID NO:1 is the DNA sequence dna of the lipase for such as from flash of light palpus mould (Phycomyces nitens) separate.
SEQ ID NO:2 is such as from SEQ ID NO:The amino acid sequence of 1 lipase for deriving.
SEQ ID NO:3 are directed to the SEQ ID NO in aspergillus oryzae:The artificial DNA sequence that 2 expression is optimized.
SEQ ID NO:4 is primer lip47921-4O_C505_BamHI.
SEQ ID NO:5 is primer lip47921-4O_C505_XhoI.
Definition
Allele variant：Term " allele variant " mean to occupy two kinds of the gene of same chromogene seat or Any one in more kinds of alternative forms.Allelic variation is naturally-produced by being mutated, and can cause polymorphic in colony Property.Gene mutation can be that silence (without change in coded polypeptide) or codified have the amino acid sequence for changing Polypeptide.The allele variant of polypeptide is by the polypeptide of the allelic variants code of gene.
cDNA：Term " cDNA " means to divide by from ripe, montage the mRNA for deriving from eucaryon or prokaryotic The DNA molecular that son carries out reverse transcription and prepares.CDNA lacks the intron sequences that may reside in correspondence genomic DNA.It is early First Initial R NA transcripts are the precursors of mRNA, and it will be through a series of step before the mRNA of montage of maturation is rendered as It is processed, including montage.
Coded sequence：Term " coded sequence " means directly to specify a polynucleotides for the amino acid sequence of polypeptide.Compile The border of code sequence is typically determined that the open reading frame is from initiation codon (such as ATG, GTG or TTG) by open reading frame Start and terminated with terminator codon (such as TAA, TAG or TGA).Coded sequence can be genomic DNA, cDNA, synthetic DNA Or its combination.
Control sequence：Term " control sequence " means that the polynucleotides for encoding mature polypeptide of the invention for expression are musted The nucleotide sequence for needing.Each control sequence can be natural (that is, from same base relative to the polynucleotides of coded polypeptide Cause) or external source (that is, from different genes), or be relative to each other natural or external source.Such control sequence include but It is not limited to conductor, Polyadenylation sequences, propeptide sequence, promoter, signal peptide sequence and transcription terminator.At least, control Sequence processed includes promoter and transcription and translation termination signal.Be conducive to these control sequences and coded polypeptide for introducing Polynucleotides code area connection specific restriction enzyme enzyme site purpose, these control sequences can be provided with multiple and connect Head.
Expression：Term " expression " includes any step for being related to polypeptide to produce, and including but not limited to, is repaiied after transcription, transcription Decorations, translation, posttranslational modification and secretion.
Expression vector：Term " expression vector " means linear or ring-shaped DNA molecule, multinuclear of the molecule comprising coded polypeptide The control sequence of thuja acid and the polynucleotides operationally with offer for its expression is connected.
Fragment：Term " fragment " mean the mature polypeptide amino (N-) and/or carboxyl (C-) terminal deletion one or A kind of polypeptide of multiple (for example, several) amino acid；Wherein the fragment has lipase active.On the one hand, the fragment bag The NO of ID containing SEQ:The number of 2 amino acid/11 to 340 at least 50%, at least 55%, at least 60%, at least 65%, at least 70%th, at least 80%, at least 85%, at least 90% or at least 95%.
Host cell：Term " host cell " means to be easy to nucleic acid construct or table comprising polynucleotides of the invention Up to any cell type of carrier conversion, transfection, transduction etc..The mutation that term " host cell " occurs during covering due to duplication And the spawn of the parental cell different from parental cell.
Separate：Term " separation " means in non-existent form in nature or the material in environment.Separate The non-limiting examples of material include (1) any non-naturally occurring material, and (2) include but is not limited to any enzyme, polypeptide, change Any material of body, nucleic acid, protein, peptide or co-factor, the material at least in part from one kind substantially related to it or Removed in various (for example, some) or all naturally occurring compositions；(3) relative to the natural material for finding by manually modified Any material；Or (4) are any by what is modified relative to the amount for increasing the material with its other components being naturally associated Material is (for example, encode multiple copies of the gene of the material；Than with the natural promoter being associated of the gene for encoding the material more The use of strong promoter).The material of separation may reside in fermentation broth sample.
Lipase：Term " lipase (lipase) ", " lipase (lipase enzyme) ", " lipolytic enzyme ", " lipid esters Enzyme ", " steatolysis polypeptide " and " steatolysis albumen " refer to a kind of enzyme in the EC 3.1.1 classes as defined in enzyme nomenclature.It can be with With lipase active (triacylglycerol lipase, EC 3.1.1.3), Cutinase activity (EC 3.1.1.74), sterol esterase Active (EC 3.1.1.13) and/or wax-ester hydrolase activity (EC 3.1.1.50).For purposes of the present invention, according in example Described program determines lipase active.On the one hand, variant of the invention has SEQ ID NO:2 polypeptide is at least 20%, for example, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%th, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100% lipase activity Property.
Low temperature：" low temperature " is 5 DEG C -35 DEG C, such as 5 DEG C -30 DEG C, 5 DEG C -25 DEG C, 5 DEG C -20 DEG C, 5 DEG C -15 DEG C or 5 DEG C -10 DEG C temperature.In another embodiment, " low temperature " is 10 DEG C -35 DEG C, such as 10 DEG C -30 DEG C, 10 DEG C -25 DEG C, 10 DEG C -20 DEG C, Or 10 DEG C -15 DEG C of temperature.
Mature polypeptide：Term " mature polypeptide " means in its final form after translation and any posttranslational modification Polypeptide, the modification such as processing of N- ends, C- ends are blocked, glycosylated, phosphorylation etc..On the one hand, mature polypeptide is SEQ ID NO:2 amino acid/11 is to 340.SEQ ID NO:2 amino acid -23 is to 1 being signal peptide.It is known in the art that place Chief cell can produce the different mature polypeptides of two or more expressed by identical polynucleotides (that is, different with one C- ends and/or -terminal amino acid) mixture.
Mature polypeptide encoded sequence：Term " mature polypeptide encoded sequence " means that maturation of the coding with lipase active is more The polynucleotides of peptide.On the one hand, the mature polypeptide encoded sequence is included in SEQ ID NO:1 nucleotides 570-1029, 1090-1118、1188-1245、1314-1410、1482-1579、1641-1727、1781-1861、1933-2011、2068- In 2101.SEQ ID NO:1 encoded signal peptide of nucleotides 501 to 569.SEQ ID NO:DNA sequence dna in 1 is derived from flash of light The mould genomic DNA of palpus.
On the one hand, mature polypeptide encoded sequence is included in SEQ ID NO:In 3 nucleotides 70-1089.SEQ ID NO:1 encoded signal peptide of nucleotides 3 to 69.In SEQ ID NO:DNA sequence dna in 3 is directed to the SEQ ID in aspergillus oryzae NO:The artificial DNA sequence that 2 expression is optimized.
Nucleic acid construct：Term " nucleic acid construct " means single-stranded or double-stranded nucleic acid molecules, and the nucleic acid molecules are from day Separated in the gene for so existing, or the section containing nucleic acid is modified in the way of being not present in nature originally, or It is synthesis, the nucleic acid molecules include one or more control sequences.
It is operably connected：Term " being operably connected " means following construction, wherein, control sequence is relative to multinuclear The coded sequence of thuja acid is disposed in position, so that the control sequence instructs the expression of the coded sequence.
Parent or parent lipase：Term " parent " or " parent lipase " mean following lipase, to the lipase Replaced to produce the variant of lipase of the invention.Parent can be naturally occurring (wild type) polypeptide or its variant or Fragment.
Sequence identity：Described with parameter " sequence identity " between two amino acid sequences or two nucleotide sequences Between correlation.
For purposes of the present invention, using such as in EMBOSS bags (EMBOSS：European Molecular Biology Open software suite, Rice (Rice) et al., 2000, science of heredity trend (Trends Genet.) 16:276-277) (preferably 5.0.0 editions or more new edition This) Maimonides your program in implemented Ned Coleman-wunsch algorithm (Ned Coleman (Needleman) and wunsch (Wunsch), 1970, J. Mol. BioL (J.Mol.Biol.) 48:It is 443-453) consistent to determine the sequence between two amino acid sequences Property.The parameter for being used be Gap Opening Penalty 10, gap extension penalties 0.5, and EBLOSUM62 (BLOSUM62's EMBOSS editions) substitution matrix.The output (use-non-reduced option is obtained) of " uniformity most long " of Maimonides that mark is used as Percent Identity, and be calculated as follows：
(consistent residue x 100)/(comparing the room sum in length-comparison)
For purposes of the present invention, using such as in EMBOSS bags (EMBOSS：European Molecular Biology Open software suite, Rice (Rice) et al., 2000, sees above) Maimonides implemented in the Maimonides of (preferably 5.0.0 editions or more redaction) your program Germania-wunsch algorithm (Ned Coleman (Needleman) and wunsch (Wunsch), 1970, see above) determines two deoxidation cores Sequence identity between ribotide sequence.The parameter for being used is Gap Opening Penalty 10, gap extension penalties 0.5, with And EDNAFULL (EMBOSS editions of NCBI NUC4.4) substitution matrix.The output of " uniformity most long " of Maimonides that mark (makes With-non-reduced option acquisition) it is used as Percent Identity, and be calculated as follows：
(consistent deoxyribonucleotide x 100)/(comparing the room sum in length-comparison)
Stringent condition：Unusual low stringency condition：Term " very low stringency condition " means that for length be at least 100 cores For the probe of thuja acid, it then follows standard DNA western blot procedure, in 5X SSPE, 0.3%SDS, 200 micrograms/mL shearings at 42 DEG C And prehybridization and hybridization 12 to 24 hours in the salmon sperm dna and 25% formamide of denaturation.It is last that 2X is used at 45 DEG C SSC, 0.2%SDS wash carrier material three times, every time 15 minutes.Low stringency condition：Term " low stringency condition " mean for For length is at least 100 probes of nucleotides, it then follows standard DNA western blot procedure, in 5X SSPE, 0.3% at 42 DEG C Prehybridization and hybridization 12 to 24 hours in salmon sperm dna that SDS, 200 micrograms/mL are sheared and is denatured and 25% formamide.Most Carrier material is washed three times, every time 15 minutes using 2X SSC, 0.2%SDS at 50 DEG C afterwards.Middle stringent condition：Term " in it is tight Glazing bar part " means for length is at least 100 probes of nucleotides, it then follows standard DNA western blot procedure, at 42 DEG C Prehybridization and hybridization in salmon sperm dna that 5X SSPE, 0.3%SDS, 200 micrograms/mL are sheared and is denatured and 35% formamide 12 to 24 hours.It is last to be washed carrier material three times, every time 15 minutes using 2X SSC, 0.2%SDS at 55 DEG C.In-Gao Yan Glazing bar part：Term " in-high stringency conditions " mean for length is at least 100 probes of nucleotides, it then follows standard DNA Western blot procedure, the salmon sperm dna and 35% sheared and be denatured in 5X SSPE, 0.3%SDS, 200 micrograms/mL at 42 DEG C Prehybridization and hybridization 12 to 24 hours in formamide.It is last that carrier material is washed three using 2X SSC, 0.2%SDS at 60 DEG C It is secondary, 15 minutes every time.High stringency conditions：Term " high stringency conditions " means that for length be at least 100 probes of nucleotides For, it then follows standard DNA western blot procedure, the salmon sheared and be denatured in 5X SSPE, 0.3%SDS, 200 micrograms/mL at 42 DEG C Prehybridization and hybridization 12 to 24 hours in fish sperm DNA and 50% formamide.Finally will using 2X SSC, 0.2%SDS at 65 DEG C Carrier material is washed three times, every time 15 minutes.Unusual high stringency conditions：Term " very high stringency conditions " means For at least 100 probes of nucleotides, it then follows standard DNA western blot procedure, in 5X SSPE, 0.3%SDS, 200 at 42 DEG C Prehybridization and hybridization 12 to 24 hours in salmon sperm dna that microgram/mL is sheared and is denatured and 50% formamide.Finally at 70 DEG C Carrier material is washed three times, every time 15 minutes using 2X SSC, 0.2%SDS.
Subsequence：Term " subsequence " means to make one or more (for example, several) nucleotides from mature polypeptide encoded The 5' ends of sequence and/or the polynucleotides of 3' ends missing；Wherein subsequence coding has the fragment of lipase active.In a side Face, sub-series of packets ID containing SEQ NO:The number of 2 nucleotides 82 to 1134 at least 50%, at least 55%, at least 60%, extremely Few 65%, at least 70%, at least 80%, at least 85%, at least 90% and at least 95%.
Variant：Term " variant " mean at one or more (for example, several) position include substitution with lipase The polypeptide of activity, i.e., variant of the invention is also polypeptide of the invention.Substitution means to take one with different amino acid replacements The amino acid of position.These variants of the invention have SEQ ID NO:At least the 20% of 2 lipase active, for example, at least 40%th, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100%.
Scourability：In the context of the present invention, removed as enzyme using term " scourability " and be present in thing to be cleaned Lipid on body or the ability containing lipid spot.Scourability can by calculate the AMSA in following methods part illustrate in So-called G/ (B+R) values of definition are quantified.Term " scourability " includes generally cleaning such as hard-surface cleaning, is such as washed in tableware In washing, but the scourability on textile such as clothing is additionally included in, and also including industry cleaning and mechanism's cleaning.
Wild type lipase：Term " wild type " lipase means (to be sent out such as in nature by naturally occurring microorganism Existing bacterium, yeast or filamentous fungi) expression lipase." wild type " lipase can carry out restructuring table in host cell Reach.
Detailed description of the invention
Polypeptide with lipase active
In one embodiment, the present invention relates to the polypeptide of the separation with lipase active, the polypeptide choosing that these are separate From the following group, the group is made up of the following：(a) and SEQ ID NO:2 mature polypeptide have at least 60%, at least 65%, extremely Few 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%th, the polypeptide of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity；(b) By the polypeptide of following polynucleotide encoding, the polynucleotides under low stringency condition, under middle stringent condition, in-high stringency conditions Under, under high stringency conditions or very under high stringency conditions with (i) SEQ ID NO:1 or SEQ ID NO:3 or (i) total length it is complementary Body hybridizes；A kind of (c) polypeptide, the polypeptide by with SEQ ID NO:1 or SEQ ID NO:3 mature polypeptide encoded sequence has extremely Few 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%th, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence The polynucleotide encoding of row uniformity；D a kind of () polypeptide, the polypeptide is included at one or more (for example, a number) position The SEQ ID NO of substitution, missing, and/or insertion:2 variant；And a kind of (e) polypeptide, the polypeptide be (a), (b), (c) or The fragment of any one of the polypeptide of (d).
In a specific embodiment, the present invention relates to SEQ ID NO:2 mature polypeptide have at least 60%, extremely Few 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence is consistent The polypeptide of property, and wherein the polypeptide has SEQ ID NO:The lipase active of at least the 70% of 2 mature polypeptide.
In a specific embodiment, the present invention relates to SEQ ID NO:2 mature polypeptide have at least 60%, extremely Few 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence is consistent The polypeptide of property, and wherein the polypeptide has SEQ ID NO:The lipase active of at least the 75% of 2 mature polypeptide.
In a specific embodiment, the present invention relates to SEQ ID NO:2 mature polypeptide have at least 60%, extremely Few 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence is consistent The polypeptide of property, and wherein the polypeptide has SEQ ID NO:The lipase active of at least the 80% of 2 mature polypeptide.
In a specific embodiment, the present invention relates to SEQ ID NO:2 mature polypeptide have at least 60%, extremely Few 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence is consistent The polypeptide of property, and wherein the polypeptide has SEQ ID NO:The lipase active of at least the 85% of 2 mature polypeptide.
In a specific embodiment, the present invention relates to SEQ ID NO:2 mature polypeptide have at least 60%, extremely Few 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence is consistent The polypeptide of property, and wherein the polypeptide has SEQ ID NO:The lipase active of at least the 90% of 2 mature polypeptide.
In a specific embodiment, the present invention relates to SEQ ID NO:2 mature polypeptide have at least 60%, extremely Few 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence is consistent The polypeptide of property, and wherein the polypeptide has SEQ ID NO:The lipase active of at least the 95% of 2 mature polypeptide.
In a specific embodiment, the present invention relates to SEQ ID NO:2 mature polypeptide have at least 60%, extremely Few 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence is consistent The polypeptide of property, and wherein the polypeptide has SEQ ID NO:The lipase active of at least the 100% of 2 mature polypeptide.
Polypeptide of the invention preferably includes SEQ ID NO:2 amino acid sequence or its allele variant or by its group Into；Or it has the fragment of lipase active.On the other hand, the polypeptide includes SEQ ID NO:2 mature polypeptide or by Its composition.On the other hand, the polypeptide includes SEQ ID NO:2 amino acid/11 is to 340 or is made from it.
In another embodiment, the present invention relates to the separation with lipase active by following polynucleotide encoding Polypeptide, the polynucleotides very low stringency condition, low stringency condition, middle stringent condition, in-high stringency conditions, strict bar high Hybridize with the following under part or very high stringency conditions：(i)SEQ ID NO:1 or SEQ ID NO:3 mature polypeptide encoded Sequence, or (i) total length complement (Pehanorm Brooker (Sambrook) et al., 1989, Molecular Cloning:A Laboratory guide (Molecular Cloning, A Laboratory Manual), the second edition, Cold SpringHarbor (Cold Spring Harbor), knob About).
On the other hand, the polypeptide is SEQ ID NO:The fragment of 2 polypeptide.The fragment can include at least 250 ammonia Base acid residue, for example, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, extremely Few 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320 Individual, at least 325, at least 330, at least 335, at least 340 amino acid residues.On the one hand, the fragment includes SEQ ID NO:The number of the amino acid of 2 polypeptide at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, extremely Few 75%, at least 80%, at least 85%, at least 90% or at least 95%.
SEQ ID NO:1 or SEQ ID NO:3 polynucleotides or its subsequence, together with SEQ ID NO:2 polypeptide or Its fragment can be used to design nucleic acid probe to identify and clone from the strain not belonged to together or plant according to method well known in the art , coding with lipase active polypeptide DNA.Specifically, standard DNA western blot procedure can be followed, such probe is used Genomic DNA or cDNA with cell interested hybridize, to be identified and isolated from correspondence gene therein.Such probe can To be significantly shorter than whole sequence, but length should be at least 15, at least for example, at least 25,35 or at least 70 nucleosides Acid.Preferably, the length of nucleic acid probe is at least 100 nucleotides, such as length be at least 200 nucleotides, at least 300 Nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides or at least 900 nucleotides.Both DNA and rna probe can be used.Typically probe is marked, For detect corresponding gene (for example, with32P、3H、35S, biotin or avidin).The present invention covers such probe.
The DNA for hybridizing with probe described above and encoding the polypeptide with lipase active can be directed to, to from this The genomic DNA or cDNA library of class other bacterial strains preparation are screened.Genomic DNA from such other bacterial strains or other DNA can be by agarose or polyacrylamide gel electrophoresis, or other isolation technics are separated.DNA from library or point From DNA can be transferred to and be fixed on nitrocellulose or other suitable carrier materials on.In order to identify and SEQ ID NO:1 Or SEQ ID NO:3 or its subsequence hybridization clone or DNA, by carrier material be used for southern blotting technique in.
For purposes of the present invention, hybridization represents the nucleic acid probe hybridization of polynucleotides and the mark for corresponding to following item： (i)SEQ ID NO:1 or SEQ ID NO:3；(ii)SEQ ID NO:1 or SEQ ID NO:3 mature polypeptide encoded sequence； (iii) its total length complement；Or (iv) its subsequence；Hybridization be it is non-be frequently as low as very high stringency conditions under carry out.At these Under the conditions of can be detected using such as x-ray film or as known in the art any other with the molecule of the nucleic acid probe hybridization Means are detected.
On the one hand, the nucleic acid probe is SEQ ID NO:1 or SEQ ID NO:3.On the other hand, the nucleic acid probe by SEQ ID NO:1 or SEQ ID NO:3 at least 15 and up to 1000 nucleotides compositions.In another aspect, nucleic acid Probe is the polynucleotides for encoding following item：SEQ ID NO:2 polypeptide；Or its fragment.
In another embodiment, the present invention relates to a kind of polypeptide of separation, the polypeptide has lipase active, by with SEQ ID NO:1 or SEQ ID NO:3 mature polypeptide encoded sequence have at least 60%, at least 65%, at least 70%, at least 75%th, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, The polynucleotide encoding of at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.
In another embodiment, the present invention relates to SEQ ID NO:2 polypeptide, SEQ ID NO:2 mature polypeptide or The variant of its fragment, these variants include substitution at one or more (for example, a number) position.In one embodiment, to SEQ ID NO:The number of the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor introduced in 2 polypeptide is 1-50,1-40,1-30,1-20,1-10 or 1-5, such as 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、 29th, 30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 or 50.
The change of these amino acid can have small property, i.e. will not interfere significantly on folding and/or the activity of protein Conserved amino acid substitution or insert；The typically 1-30 small missing of amino acid；Small is amino terminal or carboxyl terminal Extend, such as amino terminal methionine residues；The up to 20-25 small joint peptide of residue；Or by changing net charge or another One function is conducive to the small extension of purifying, such as polyhistidyl section, epitope or binding structural domain.
The example of conservative replacement is in the range of the following group：Basic amino acid (arginine, lysine and histidine), acidity Amino acid (glutamic acid and aspartic acid), polar amino acid (glutamine and asparagine), hydrophobic amino acid (leucine, Isoleucine and valine), aromatic amino acid (phenylalanine, tryptophan and tyrosine) and p1 amino acid (glycine, the third ammonia Acid, serine, threonine and methionine).The 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor that specific activity will not typically be changed be it is known in the art and For example by H. Neuraths (Neurath) and R.L. Xi Er (Hill), 1979, at protein (The Proteins), science goes out Version society (Academic Press), described in New York.Common substitution be Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Tyr/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/ Ile, Leu/Val, Ala/Glu and Asp/Gly.
Alternately, amino acid change has the quality that：Change the physicochemical characteristics of polypeptide.For example, amino acid Change can improve the heat endurance of polypeptide, change substrate specificity, changes optimal pH etc..
Can be according to program as known in the art, such as direct mutagenesis or alanine scanning mutagenesis (Kan Ninghan (Cunningham) and Weir this (Wells), 1989, science (Science) 244:1081-1085) come identify in polypeptide must Need amino acid.In rear kind of technology, single alanine mutation is introduced at each residue in the molecule, and test gained mutation The lipase active of molecule is identifying the amino acid residue crucial to the activity of molecule.Referring also to Hilton (Hilton) et al., 1996, journal of biological chemistry (J.Biol.Chem.) 271:4699-4708.Also can combine and assume the prominent of contact site amino acids Become, be such as determined by following technology such as nuclear magnetic resonance, crystallography, electronic diffraction or photoaffinity labeling, structure is carried out Physics analysis, so that it is determined that the avtive spot of enzyme or other biological interact.See, e.g., De Wosi (de Vos) Et al., 1992, science (Science) 255:306-312；Smith (Smith) et al., 1992, J. Mol. BioL (J.Mol.Biol.)224:899-904；Waller Da Er (Wlodaver) et al., 1992, FEBS's communication (FEBS Lett.)309:59-64.Can also infer from the comparison with related polypeptide and differentiate essential amino acid.
Single or multiple 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors can be made, is lacked and/or is inserted and use mutagenesis, restructuring, and/or reorganization Known method tested, then carry out related screening sequence, such as by Reed Ha Er-Mancur Olson (Reidhaar-Olson) and Sa Aoer (Sauer), 1988, science (Science) 241:53-57；Bo Wei (Bowie) and Sa Aoer, 1989, American National section Institute's proceeding (Proc.Natl.Acad.Sci.USA) 86:2152-2156；WO 95/17413；Or the disclosures of WO 95/22625 Those.The method that other can be used includes fallibility PCR, phage display (such as Lip river graceful (Lowman) et al., 1991, biology Chemistry (Biochemistry) 30:10832-10837；US 5223409；WO 92/06204) and regiondirected mutagenesis (Derby Shi Er (Derbyshire) et al., 1986, gene (Gene) 46:145；Nellie (Ner) et al., 1988, DNA 7:127).
Can be detected with high throughput automated screening technique by the clone of host cell expression with combined mutagenesis/Shuffling Method , activity (interior this (Ness) et al., 1999, Nature Biotechnol (Nature Biotechnology) 17 of the polypeptide of mutagenesis: 893-896).The DNA molecular of the mutagenesis of encoding active polypeptide can be recovered from host cell, and use the standard side of this area Method is sequenced rapidly to it.These methods allow the rapid importance for determining single amino acids residue in polypeptide.
Polypeptide can be hybrid polypeptide, and the region of one of polypeptide is merged at N-terminal or the C end in the region of another polypeptide End.
Polypeptide can also be fused polypeptide or cleavable fused polypeptide, wherein another peptide fusion is in polypeptide of the invention N- ends or C- ends.Fusion is produced by the way that the polynucleotides for encoding another polypeptide are fused into polynucleotides of the invention Polypeptide.Technology for producing fused polypeptide is known in the art, and including connecting the coded sequence of coded polypeptide, this Sample causes them in inframe and causes control of the expression of fused polypeptide in one or more promoters of identical and terminator Under system.Fused polypeptide can also be built using intein technology, and wherein fused polypeptide produces (cooper (Cooper) upon translation Et al., 1993, European Molecular Bioglogy Organization's magazine (EMBO J.) 12:2575-2583；Road gloomy (Dawson) et al., 1994, Science (Science) 266:776-779).
Fused polypeptide can further include cleavage site between two kinds of polypeptides.When fusion protein is secreted, the position Point is cut, so as to discharge both polypeptides.The example of cleavage site includes but is not limited to the site disclosed in the following： Martin (Martin) et al., 2003, industrial microbiology and biotechnology magazine (J.Ind.Microbiol.Biotechnol.) 3：568-576；Si Weidina (Svetina) et al., 2000, biotechnology magazine (J.Biotechnol.) 76：245-251；Draw Si Masen (Rasmussen)-Wilson's (Wilson) et al., 1997, using with environmental microbiology (Appl.Environ.Microbiol.)63：3488-3493；Hua De (Ward) et al., 1995, biotechnology (Biotechnology)13：498-503；And hole Te Lasi (Contreras) et al., 1991, biotechnology 9：378-381； Eton (Eaton) et al., 1986, biochemistry (Biochemistry) 25：505-512；Collins (Collins)-Lai Si (Racie) et al., 1995, biotechnology 13：982-987；Ka Te (Carter) et al., 1989, albumen：Structure, function and something lost Pass and learn (Proteins:Structure,Function,and Genetics)6：240-248；And Glenn Stevens (Stevens), 2003, international drugs find (Drug Discovery World) 4：35-48.
Can also be the one or more (examples for producing lipase Variant in view of polypeptide of the invention as described above Such as several) substitution offer basis.Therefore, the polypeptide should also be parent lipase.
The source of the polypeptide with lipase active
Polypeptide with lipase active of the invention can be obtained from the microorganism of any category.For mesh of the invention , a kind of such as use with reference to given source herein term " from ... middle acquisition " should mean by the polypeptide of polynucleotide encoding It is to be produced by the source or by the bacterial strain for being wherein already inserted into the polynucleotides from the source.On the one hand, the polypeptide It is exocytosis.
The polypeptide can be comprising lipase of bacterial origin or fungal lipase.At a preferred aspect, the polypeptide is that flash of light must be mould Lipase, for example, SEQ ID NO:2 lipase, SEQ ID NO:2 mature polypeptide or its fragment.
It will be appreciated that for above-mentioned species, the present invention covers complete state and partial state (perfect and imperfect states) the two and other taxology equivalents, such as phorozoon, but regardless of them What known species name is.Those of ordinary skill in the art will readily recognize the identity of appropriate equivalent.
The bacterial strain of bacterium and fungal species can be easily for the public to obtain in many culture collections, such as the U.S. Type Tissue Collection (ATCC), Germany Microbiological Culture Collection Center (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), Centraalbureau collection (Centraalbureau Voor Schimmelcultures, CBS) and american agriculture research Service Patent Culture collection northern area research Center (NRRL).
Can be originated from other using above-mentioned probe, including dividing from nature (for example, soil, compost, water etc.) From microorganism or the DNA sample identification that is directly obtained from nature material (for example, soil, compost, water etc.) and obtain the polypeptide. Technology for being directly separated microorganism and DNA from natural living environment is well known in the art.Then can be by another Similarly screened to obtain many of coded polypeptide in the genomic DNA or cDNA library of planting microorganism or hybrid dna sample Nucleotides.Once with the polynucleotides of one or more probe in detecting to coded polypeptide, it is possible to common by using this area Technology known to technical staff separate or clone the polynucleotides (see, for example, Pehanorm Brooker (Sambrook) et al., 1989, ibid).
The preparation of polypeptide
Method the invention further relates to be used to obtain the polypeptide with lipase active, these methods include：A () is to parent Correspond to SEQ ID NO in lipase (for example, wild type):One or more (for example, several) positions of the position of 2 polypeptide Put place and introduce substitution；And (b) reclaims the variant.
These variants, such as direct mutagenesis, synthetic gene can be prepared using any mutagenesis procedures known in the art Structure, semi-synthetic gene constructed, random mutagenesis, reorganization etc..
Direct mutagenesis is that one or more the restriction sites in the polynucleotides for encoding the parent introduce one or many The technology of individual (for example, several) mutation.
Direct mutagenesis can be in vitro realized by using the PCR of the Oligonucleolide primers containing desired mutation is related to. Site direct mutagenesis can also be carried out by cassette mutagenesis, the cassette mutagenesis is related to by restriction enzyme including many of coding parent Site cutting and the subsequent oligonucleotides that will contain mutation in the plasmid of nucleotides will be connected in polynucleotides.Generally, The restriction enzyme for digesting the plasmid and the oligonucleotides is identical, with allow the plasmid cohesive end and Insert Fragment each other Connection.See, e.g. and thank Le (Scherer) and Davis (Davis), 1979, NAS's proceeding (Proc.Natl.Acad.Sci.USA)76:4949-4955；With bar (Barton) et al., 1990, nucleic acids research (Nucleic Acids Res.)18:7349-4966。
Can also be by realizing direct mutagenesis in methods known in the art body.See, e.g., US 2004/0171154； Tim Story uncommon (Storici) et al., 2001, Nature Biotechnol (Nature Biotechnol.) 19:773-776；Card human relations (Kren) et al., 1998, Natural medicine (Nat.Med.) 4:285-290；And Kai Lisanuo (Calissano) and Maqino (Macino), 1996, Fungal Genetics news in brief (Fungal Genet.Newslett.) 43:15-16.
Any direct mutagenesis program can be used in the present invention.In the presence of many commercially available examination that can be used to prepare variant Agent box.
Synthetic gene builds that to need to synthesize in vitro a kind of polynucleotide molecule of design a kind of interested more to encode Peptide.Gene chemical synthesis can be carried out using multiple technologies, such as by field (Tian) et al. (2004, natural (Nature) 432:1050- 1054) technology based on multichannel microchip described in and wherein synthesize on the programmable micro flow chip of light and assemble few core The similar techniques of thuja acid.
Using known mutagenesis, restructuring and/or Shuffling Method, then carrying out a screening sequence for correlation can make list One or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, missing and/or insertion are simultaneously tested it, and these related screening sequences are for example by Rui Deha That-Mancur Olson (Reidhaar-Olson) and Sa Aoer (Sauer), 1988, science 241:53-57；Bao Yi (Bowie) and Sa are difficult to understand You, 1989, NAS's proceeding (Proc.Natl.Acad.Sci.USA) 86:2152-2156；WO 95/17413；Or WO 95/22625.The method that other can be used include fallibility PCR, phage display (such as Lip river graceful (Lowman) et al., 1991, biochemistry (Biochemistry) 30:10832-10837；US 5,223,409；WO 92/06204) and region it is fixed To mutagenesis (Derby Shi Er (Derbyshire) et al., 1986, gene (Gene) 46:145；Nellie (Ner) et al., 1988, DNA 7:127)。
Can be detected with high throughput automated screening technique by the clone of host cell expression with combined mutagenesis/Shuffling Method , activity (interior this (Ness) et al., 1999, Nature Biotechnol (Nature Biotechnology) 17 of the polypeptide of mutagenesis: 893-896).The DNA molecular of the mutagenesis of encoding active polypeptide can be recovered from host cell, and use the standard side of this area Method is sequenced rapidly to it.These methods allow the rapid importance for determining single amino acids residue in polypeptide.
By the way that combinatorial compound is gene constructed, and/or direct mutagenesis, and/or random mutagenesis, and/or many aspects of reorganization It is semi-synthetic gene constructed to realize.Semi-synthetic structure is typically, using the process combination PCR skills of the polynucleotide passage of synthesis Art.Therefore, the region of the restriction of gene can be with de novo formation, and other regions can be expanded using site-specific mutagenesis primer Increase, and also have other regions to undergo fallibility PCR or non-fallibilities PCR and expand.Then polynucleotides subsequence can be carried out Reorganization.Nucleic acid construct
The invention further relates to nucleic acid construct, these nucleic acid constructs are controlled comprising one or more are operably coupled to The polynucleotides of the invention of sequence, under conditions of compatible with control sequence, these control sequences instruct coded sequence closing Expression in suitable host cell.
The polynucleotides can be manipulated in many ways, to provide the expression of the polypeptide.Depending on expression vector, in multinuclear It can be desirable or required to carry out manipulation to it before thuja acid insertion vector.For using recombinant DNA method modification The technology of polynucleotides is well known in the art.
Control sequence can be promoter, i.e., recognize the polynucleotides for expressing the polynucleotides by host cell.Should Promoter includes transcriptional control sequence, and these sequences mediate the expression of the polypeptide.The promoter can be shown in host cell Any polynucleotides of transcriptional activity, including saltant type, truncated-type and hybrid promoters are shown, and by coding and can be somebody's turn to do Homologous or heterologous extracellular or intracellular polypeptides the gene of host cell is obtained.
In bacterial host cell, the example for being adapted to the promoter of guidance nucleic acid construct transcription of the invention is to be derived from The promoter of the following：Bacillus amyloliquefaciens alpha-amylase gene (amyQ), bacillus licheniformis alpha-amylase gene (amyL), bacillus licheniformis penicillinase gene (penP), bacillus stearothermophilus maltogenic amylase gene (amyM), withered Careless bacillus type froctosan saccharase gene (sacB), bacillus subtilis xylA and xylB gene, bacillus thuringiensis CryIIIA genes (A Gaisi (Agaisse) and inner Rake Lars (Lereclus), 1994, molecular microbiology (Molecular Microbiology)13:97-107), E. coli lac operon, Escherichia coli trc promoters (Ai Gong (Egon) et al., 1988, gene) 69:301-315), streptomyces coelicolor agarase gene (dagA) and protokaryon beta-lactam enzyme gene (Wella-card Ma Nuofu (Villa-Kamaroff) et al., 1978, NAS's proceeding 75:3727-3731), together with tac promoters (moral bohr (DeBoer) et al., 1983, NAS's proceeding 80:21-25).Other promoter is described in and " comes from The useful proteins (Useful proteins from recombinant bacteria) of recombinant bacteria ", gilbert (Gilbert) et al., 1980, science American (Scientific American), 242:74-94；And Pehanorm Brooker (Sambrook) et al., 1989, ibid.The example of Gene expression is disclosed in WO 99/43835.
Reality for instructing the suitable promoter of transcription of the nucleic acid construct of the invention in filamentous fungal host cell Example is the promoter obtained from the gene of the following：Aspergillus nidulans acetamidase, Aspergillus ni ger neutral AMS, aspergillus niger acid Stability AMS, aspergillus niger or Aspergillus awamori amylase (glaA), oryzae TAKA amylase, Aspergillus oryzae alkaline egg White enzyme, aspergillus oryzae triose-phosphate isomerase, Fusarium oxysporum trypsin enzyme sample protease (WO 96/00787), empiecement Fusariumsp Amyloglucosidase (WO 00/56900), empiecement Fusariumsp Daria (Fusarium venenatum Daria) (WO 00/ 56900), empiecement fusarium Quinn (Fusarium venenatum Quinn) (WO 00/56900), rhizomucor miehei (Rhizomucor miehei) lipase, rhizomucor miehei aspartic protease, trichoderma reesei β-glucosyl enzym, Richter scale wood Mould cellobiohydrolase I, trichoderma reesei cellobiohydrolase II, trichoderma reesei endoglucanase I, trichoderma reesei inscribe Dextranase II, trichoderma reesei endoglucanase III, trichoderma reesei endoglucanase IV, trichoderma reesei endo-glucanase Enzyme V, Xylanase from Trichoderma reesei I, Xylanase from Trichoderma reesei II, trichoderma reesei xylobiase, and NA2tpi promoters (promoter of modification, it comes from aspergillus neutral alpha-amylase enzyme gene, wherein untranslated targeting sequencing is by aspergillus triose phosphorus The untranslated targeting sequencing of acid isomer enzyme gene is substituted；Non-limiting examples include the promoter of modification, and it comes from aspergillus niger The gene of neutral alpha-amylase, wherein untranslated targeting sequencing is by aspergillus nidulans or aspergillus oryzae triose phosphate isomerase gene Untranslated targeting sequencing is substituted)；And its saltant type promoter, truncated-type promoter and hybrid promoters.
In yeast host, useful promoter is derived from the gene of the following：Saccharomyces cerevisiae enolase (ENO1), wine Brewer yeast galactokinase (GAL1), saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), wine Brewer yeast triose-phosphate isomerase (TPI), brewing yeast metallothionein (CUP1) and saccharomyces cerevisiae 3-phoshoglyceric acid swash Enzyme.Rome Northey (Romanos) et al., 1992, yeast (Yeast) 8:423-488 describe yeast host cell other have Promoter.
Control sequence can also be and be recognized to terminate the transcription terminator of transcription by host cell.Terminator sequence quilt can Be operably connected to encode the polypeptide polynucleotides 3 '-end.The tool functional in host cell can be used any Terminator.
Preferred terminator for bacterial host cell is from Bacillus clausii alkali protease (aprH), lichens bud The gene of spore a-Amylase Bacillus (amyL) and Escherichia coli rRNA (rrnB) is obtained.
The preferred terminator of filamentous fungal host cell is obtained from the gene of the following：Aspergillus nidulans neighbour's amino Benzoic acid synthase, aspergillus niger glucoamylase, aspergillus niger alpha-glucosidase, oryzae TAKA amylase and Fusarium oxysporum Trypsin like proteases.
Preferred terminator for yeast host cell is from saccharomyces cerevisiae enolase, S. cerevisiae cytochrome C (CYC1) and S. cerevisiae glyceraldehyde -3- phosphate dehydrogenases gene obtain.Rome Northey (Romanos) et al., 1992, Ibid, other useful terminators of yeast host cell are described.
Control sequence can also be the mRNA stabilization sub-districts of the upstream of coding sequence of promoter downstream and gene, and it increases should The expression of gene.
The example of suitable mRNA stabilization sub-districts is obtained from following：Bacillus thuringiensis cryIIIA genes (WO 94/25612) ((Hue) et al., 1995, Bacteriology (Journal of are changed with bacillus subtilis SP82 genes Bacteriology)177:3465-3471)。
The control sequence can also be targeting sequencing, the untranslated mRNA region critically important to host cell translation.Before this Guide sequence is operably coupled to 5 '-end of the polynucleotides for encoding the polypeptide.Can use and be acted as in host cell Any targeting sequencing.
Preferred targeting sequencing for filamentous fungal host cell is from oryzae TAKA amylase and aspergillus nidulans triose The gene of phosphoric acid isomerase is obtained.
The suitable conductor of yeast host cell is obtained from the gene of the following：Saccharomyces cerevisiae enolase (ENO 1), the phosphoglyceric kinase of saccharomyces cerevisiae 3, cerevisiae alpha-factor and saccharomyces cerevisiae alcohol dehydrogenase/phosphorus of glyceraldehyde -3 Acidohydrogenase (ADH2/GAP).
The control sequence can also be a kind of Polyadenylation sequences, that is, be operably connected to the polypeptid coding sequence 3 '-end and be identified as being added to polyadenosine residues on transcribed mRNA by host cell when transcription one A kind of sequence of signal.Any Polyadenylation sequences worked in host cell can be used.
The preferred Polyadenylation sequences of filamentous fungal host cell are obtained from the gene of the following：Structure nest is bent Mould anthranilate synthase, aspergillus niger glucoamylase, aspergillus niger alpha-glucosidase, oryzae TAKA amylase and point Fusarium oxysporum trypsin like proteases.
The Polyadenylation sequences useful for yeast host cell are in Guo (Guo) and thank to Germania (Sherman), and 1995, Molecular cytobiology (Mol.Cellular Biol.) 15:Described in 5983-5990.
Control sequence can also be that coding is connected to the signal peptide of the N- ends of polypeptide and and guides the polypeptide to enter cell The signal peptide coding region domain of secretory pathway.5 ' ends of the coded sequence of polynucleotides can be included in natural in translation reading frame in itself The signal coding sequence being connected with the coded sequence section of coded polypeptide.Alternately, 5 ' ends of the coded sequence can With comprising being external signal coding sequence for the coded sequence.Do not compiled comprising signal peptide natively in coded sequence In the case of code sequence, it may be necessary to foreign signal peptide coding sequence.Alternately, foreign signal peptide coding sequence can be simply Natural signal coding sequence is replaced to strengthen the secretion of the polypeptide.However, it is possible to use instructing expressed polypeptide to enter Any signal coding sequence of the secretion path of host cell.
Useful signal peptide-coding sequence for bacterial host cell is that the signal peptide obtained from the gene of the following is compiled Code sequence：Bacillus NCIB 11837 produces maltogenic amylase, bacillus licheniformis subtilopeptidase A, lichens gemma Bacillus beta-lactamase, bacillus stearothermophilus alpha-amylase, stearothermophilus neutral protease (nprT, nprS, ) and bacillus subtilis prsA nprM.Simon is received (Simonen) and Paar watt (Palva), 1993, Microbi (Microbiological Reviews)57:109-137 describes other signal peptide.
Useful signal peptide-coding sequence for filamentous fungal host cell is the letter that is obtained from the gene of the following Number peptide-coding sequence：Aspergillus ni ger neutral amylase, aspergillus niger glucoamylase, oryzae TAKA amylase, Humicola insolens are fine The plain enzyme of dimension, Humicola insolens EGV, Humicola lanuginosa lipase and rice black wool mould aspartic protease.
The signal peptide useful for yeast host cell is derived from the gene of following item：Cerevisiae alpha-factor and wine brewing ferment Female invertase.Rome Northey (Romanos) et al., 1992, ibid, describe other useful signal coding sequences.
Control sequence can also be propeptide code sequence of the coding positioned at the propetide of the N- ends of polypeptide.The polypeptide quilt of generation Referred to as preemzyme (proenzyme) or propolypeptide (or being referred to as proenzyme (zymogen) in some cases).Propolypeptide is usual It is inactive and can be many by from catalysis cutting on the propolypeptide or autocatalysis cutting propetide being converted to activity Peptide.Propeptide code sequence can be obtained from the gene of the following：Bacillus subtilis alkali proteinase (aprE), withered grass gemma Bacillus neutral proteinase (nprT), Myceliophthora thermophila laccase (WO 95/33836), Man Hegen Mucors aspartic protease, with And cerevisiae alpha-factor.
When signal peptide and propeptide sequence simultaneously in the presence of, propeptide sequence is positioned next in the N- ends of polypeptide, and signal Peptide sequence is positioned next in the N- ends of propeptide sequence.
It may also be desirable to add regulatory sequence, these regulatory sequences adjust the table of polypeptide relative to the growth of host cell Reach.The example of regulating system is in response to cause those that the expression of gene is turned on and off in chemical or physical stimulus, including The presence of modulating compound.Regulating system in prokaryotic system includes lac, tac and trp operator system.In yeast In, it is possible to use ADH2 systems or GAL1 systems.In filamentous fungi, it is possible to use aspergillus niger glucoamylase promoter, rice Aspergillus TAKA AMSs promoter and aspergillus oryzae glucoamylase promoter.The other examples of regulatory sequence are to allow base Those of gene-amplification.In eukaryotic system, these regulating and controlling sequences are included in the dihydrofoilic acid being amplified in the presence of methotrexate (MTX) also Nitroreductase gene and the metallothionein gene expanded with heavy metal.In these cases, the polynucleotides for encoding the polypeptide will It is operably connected with regulating and controlling sequence.
Expression vector
The invention further relates to the restructuring comprising polynucleotides of the invention, promoter and transcription and translation termination signal Expression vector.Different nucleotides and control sequence can be linked together to produce recombinant expression carrier, and the recombination expression is carried Body can include one or more easily restriction site with allow these sites insert or substitution encode the polypeptide Polynucleotides.Alternately, the polynucleotides can be by by the polynucleotides or nucleic acid construct including the polynucleotides Body is inserted for being expressed in the suitable carrier expressed.When the expression vector is produced, the coded sequence is located in the carrier, this Sample causes that the suitable control sequence that the coded sequence is expressed with the confession is operably connected.
Recombinant expression carrier can be can advantageously undergo recombinant DNA program and can cause polynucleotides express it is any Carrier (for example, plasmid or virus).It is thin with the host for having the carrier to be introduced that the selection of carrier will typically depend on the carrier The compatibility of born of the same parents.The carrier can be linear or closure cyclic plasmid.
Carrier can be autonomously replicationg vector, i.e. used as the carrier that extrachromosomal entity is present, it is replicated independently of dyeing Body is replicated, for example, plasmid, extra-chromosomal element, minichromosomes or artificial chromosome.The carrier can be used to ensure comprising any The key element of self-replacation.Alternately, the carrier can be such carrier, when it is introduced into the host cell, be integrated Replicated in genome and together with wherein its one or more chromosomes have been incorporated.In addition it is possible to use single load Body or plasmid or two or more carriers or plasmid (these carriers or the common gene containing to be introduced into host cell of plasmid STb gene in group) or transposons.
The carrier preferably comprises one or more and allows easily to select transformed cells, transfectional cell, transducer cell etc. thin The selected marker of born of the same parents.Selected marker is such a gene, and the product of the gene provides biocide resistance or virus Resistance, heavy metal resistance, auxotrophic prototrophy etc..
The example of bacillary selected marker is bacillus licheniformis or bacillus subtilis dal genes, or assigns antibiosis The mark of plain resistance (such as ampicillin, chloramphenicol, kanamycins, neomycin, spectinomycin or tetracyclin resistance).For The suitable mark of yeast host cell includes but is not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.With In the selected marker used in filamentous fungal host cell including but not limited to amdS (acetamidase), argB (ornithines Carbamylrtansferase), bar (phosphine oxamate transacetylase), hph (hygromix phosphotransferase), niaD (nitrate reductase), PyrG (orotidine -5 ' phosphate decarboxylase), sC (sulfate adenylyl transferase) and trpC (anthranilate synthase), even With its equivalent.It is preferred that using aspergillus nidulans or aspergillus oryzae amdS and pyrG gene and water suction chain in Aspergillus cell Mould bar genes.
Carrier preferably comprise permission vector integration in the genome of host cell or carrier in cell independently of gene One or more elements of group autonomous replication.
For being incorporated into the host cell gene group, the carrier can rely on encode the polypeptide polynucleotide sequence or Person is used for by any other element of homologous or non-homologous re-combination to the carrier in the genome.Alternately, should Carrier can be comprising for instructing to be incorporated into by homologous recombination in one or more chromosomes in host cell gene group One or more accurate locations other polynucleotides.In order to increase the possibility integrated in exact position, these are whole Close element should include sufficient amount of nucleic acid, such as 100 to 10,000 base-pair, 400 to 10,000 base-pair and 800 to 10,000 base-pair, these base-pairs are homologous heavy to improve with the sequence identity that corresponding target sequence has height The possibility of group.These integrated elements can be the homologous any sequence of target sequence in the genome with host cell.Additionally, These integrated elements can be non-coding polynucleotide or coded polynucleotide.On the other hand, the carrier can be by non-same Source recombination and integration is in the genome of host cell.
For autonomous replication, the carrier may further include enables the carrier autonomous in the host cell for being discussed The replication orgin of duplication.Replication orgin can be any plasmid replicon of the mediation autonomous replication worked in cell.Art Language " replication orgin (origin of replication) " or " plasmid replicon (plasmid replicator) " mean so that The polynucleotides that plasmid or carrier can be replicated in vivo.
The example of the replication orgin of bacterium be allow replicated in Escherichia coli pBR322 plasmid, pUC19, PACYC177 and pACYC184 and pUB110, pE194, pTA1060 for being replicated in bacillus of permission and The replication orgin of pAM β 1.
The example of the replication orgin for being used in yeast host cell is 2 micron origin of replication ARS1, ARS4, ARS1 The combination of combination and ARS4 and CEN6 with CEN3.
In filamentous fungal cells the example of useful replication orgin be AMA1 and ANS1 (Ge Musi (Gems) et al., 1991, gene (Gene) 98:61-67；Card human relations (Cullen) et al., 1987, nucleic acids research (Nucleic Acids Res.) 15: 9163-9175；WO 00/24883).Method according to being disclosed in WO 00/24883 can realize separation and the bag of AMA1 genes The structure of plasmid or carrier containing the gene.
Can by the more than one copy Insertion Into Host Cell of polynucleotides of the invention increasing the generation of polypeptide.It is logical Cross by least one of sequence other copy be incorporated into host cell gene group or by comprising with the polynucleotides one The amplifiable selected marker for rising can obtain the increased copy number of polynucleotides, wherein by appropriate choosing Cultured cells can select cell, the Yi Jiyou of the copy through expanding comprising selected marker in the presence of selecting property reagent The other copy of this polynucleotides.
For connecting said elements to build the program of recombinant expression carrier of the invention for ordinary skill people It is well known (see, e.g. Sa and draw Brooker (Sambrook) et al., 1989, ibid) for member.
Host cell
The invention further relates to recombinant host cell, these recombinant host cells include coding polypeptide of the invention, can grasp It is connected to the polynucleotides of one or more control sequences with making, one or more control sequences instruct polypeptide of the invention Produce.To include that the construct or carrier of polynucleotides are introduced into host cell, so that the construct or carrier are maintained As chromosomal integrant or as the external carrier of the dyeing of autonomous replication, as noted earlier.Term " host cell " cover by The spawn of the mutation of the generation parental cell different from parental cell in reproduction process.The selection of host cell is very big Depend on encoding gene and its source of the polypeptide in degree.
Host cell can be useful any cell in restructuring produces polypeptide, such as prokaryotic or eukaryotic.
Prokaryotic host cell can be any Gram-positive or gramnegative bacterium.Gram-positive bacterium includes, But it is not limited to bacillus, fusobacterium, enterococcus spp, Geobacillus, lactobacillus, lactococcus, ocean gemma bar Pseudomonas (Oceanobacillus), staphylococcus, streptococcus and streptomyces.Gramnegative bacterium includes, but does not limit In campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacterium, mud Bacillus, neisseria, pseudomonad Category, Salmonella and Ureaplasma.
Bacterial host cell can be the cell of any bacillus, including but not limited to Alkaliphilic bacillus, solution starch It is bacillus, bacillus brevis, Bacillus circulans, Bacillus clausii, bacillus coagulans, bacillus firmus, bright Rotten bacillus, bacillus lentus, bacillus licheniformis, bacillus megaterium, bacillus pumilus, stearothermophilus gemma bar The cell of bacterium, bacillus subtilis and bacillus thuringiensis.
Bacterial host cell can also be the cell of any streptococcus, including but not limited to streptococcus equisimilis, wine purulence chain The cell of coccus, streptococcus uberis and zooepidemicus.
Bacterial host cell can also be any streptomyces cell, including but not limited to not streptomyces chromogenes, deinsectization chain Mould, streptomyces coelicolor, streptomyces griseus and shallow Streptomyces glaucoviolaceus cell.
DNA is introduced into bacillus cell can be realized by following：Protoplast transformation (see, for example, and open (Chang) and Koln (Cohen), 1979, molecular genetics and genomics (Mol.Gen.Genet.) 168:111-115), feel (poplar lattice (Young) and Spizien (Spizizen), 1961, Bacteriology are see, e.g., by state cell transformation (J.Bacteriol.)81:823-829；Or Du Bainu (Dubnau) and David Du Fu-Abbe Ademilson (Davidoff- Abelson), 1971, J. Mol. BioL (J.Mol.Biol.) 56:209-221), electroporation (see, e.g., Mao Chuan (Shigekawa) and dongle (Dower), 1988, biotechnology (Biotechniques) 6:742-751) or engagement (ginseng See, for example gram Le (Koehler) and Sohne (Thorne), 1987, Bacteriology (J.Bacteriol.) 169:5271- 5278).The Hana Chinese (Hanahan), 1983, J. Mol. BioL (see, e.g., by protoplast transformation (J.Mol.Biol.)166:557-580) or electroporation (see, e.g., dongle (Dower) et al., 1988, nucleic acids research (Nucleic Acids Res.)16:Can 6127-6145) realize being incorporated into DNA in Bacillus coli cells.DNA is introduced Can be realized by following in Streptomyces cell：Protoplast transformation, electroporation (see, e.g., tribute (Gong) et al., 2004, the linear microbiology of leaf (Folia Microbiol.) (Praha (Prague)) 49:399-405), engagement is (referring to example Such as, Ma Zuodiye (Mazodier) et al., 1989, Bacteriology (J.Bacteriol.) 171:3583-3585) or transduction (see, e.g., Bai Ke (Burke) et al., 2001, PNAS (Proc.Natl.Acad.Sci.USA) 98: 6289-6294).DNA is introduced into pseudomonas cell can be realized by following：Electroporation (see, e.g., Cai (Choi) et al., 2006, micro-biological process magazine (J.Microbiol.Methods) 64:391-397) or engagement (referring to, For example, intracutaneous many (Pinedo) and Si Meici (Smets), 2005, using with environmental microbiology (Appl.Environ.Microbiol.)71:51-57).DNA is introduced into streptococcus cell can be realized by following：My god Right competence (see, e.g., Perry (Perry) He Zangman (Kuramitsu), 1981, infection and immune (Infect.Immun.)32：1295-1297), protoplast transformation (see, e.g., Kate (Catt) and Qiao Like (Jollick), 1991, microbiology (Microbios) 68:189-207), electroporation (see, e.g., Bark profit (Buckley) et al., 1999, using with environmental microbiology (Appl.Environ.Microbiol.) 65:3800-3804)、 Or engagement (see, e.g., Ke Laiweier (Clewell), 1981, Microbi (Microbiol.Rev.) 45：409- 436).However, it is possible to use any method for being introduced into DNA in host cell known in the art.
Host cell can also be eukaryotic, such as mammal, insect, plant or fungal cell.
Host cell can be fungal cell." fungi " includes Ascomycota (Ascomycota), load as used herein Daughter bacteria door (Basidiomycota), chytridiomycota (Chytridiomycota) and Zygomycota (Zygomycota), together with ovum Bacterium door (Oomycota) and whole mitosporic fungis are (such as by Hawkesworth (Hawksworth) et al. in Ainsworth With visit this than fungi dictionary (Ainsworth and Bisby ' s Dictionary of The Fungi), the 8th edition, 1995, state Border application in biological science center (CAB International), university press (University Press), Britain Camb It is defined in (Cambridge, UK)).
The fungal host cells can be yeast cells." yeast " includes ascosporogenous yeast (endomyces as used herein Mesh), basidiosporogenous yeast and belong to the yeast of Fungi Imperfecti (gemma guiding principle).Because being sorted in for yeast may change in the future, go out In the purpose of the present invention, yeast should be such as biology of yeast and activity (Biology and Activities of Yeast) (this Jenner (Skinner), Pasmore (Passmore) and Davenport (Davenport) editor, SAB begs for By meeting (Soc.App.Bacteriol.Symposium) the 9th phase of series, 1980) it is defined describedly.
Yeast host cell can be candida, Hansenula, Saccharomyces kluyveri category, pichia, yeast Category, Schizosaccharomyces or Yarrow saccharomyces cell, such as Kluyveromyces Lactis not yeast, saccharomyces carlsbergensis (Saccharomyces Carlsbergensis), saccharomyces cerevisiae, saccharomyces diastaticus, Doug Laplace yeast (Saccharomyces douglasii), Crewe Take yeast (Saccharomyces kluyveri), promise ground enzyme mother (Saccharomyces norbensis), ellipsoideus yeast or Sub- sieve solution fat yeast (Yarrowia lipolytica) cell.
Fungal host cells can be filamentous fungal cells." filamentous fungi " includes Eumycota (Eumycota) and oomycota Subphylum (such as by Hawkesworth (Hawksworth) et al., 1995, as defined above) all filamentous forms.It is thread true Bacterium is generally characterized by what is be made up of chitin, cellulose, glucan, shitosan, mannosan and other complicated polysaccharide Mycelia body wall.Nutrient growth is extended by mycelia, and carbon catabolism is obligate aerobic.Conversely, yeast is (such as wine brewing ferment It is female) nutrient growth be that, by sprout (budding) of unicellular thallus, and carbon catabolism can be fermentation.
Filamentous fungal host cell can be acremonium, aspergillus, Aureobasidium, smoke pipe pseudomonas, plan wax Pseudomonas, gold Spore Pseudomonas, ghost agaric category, Coriolus Qu61, Cryptococcus, line black powder saccharomyces, Fusarium, Humicola, Magnaporthe grisea category, hair Mould category, myceliophthora, new U.S.'s whip Pseudomonas, Neurospora, paecilomyces, Penicillium, flat lead fungi category, white rot Pseudomonas, cud kettle Pseudomonas, Pleurotus, Schizophyllum, Talaromyces, thermophilic ascomycete category, Thielavia, Tolypocladium, Trametes or trichoderma Cell.
For example, filamentous fungal host cell can be aspergillus awamori, smelly aspergillus, aspergillus fumigatus, aspergillus japonicus, aspergillus nidulans, Aspergillus niger, aspergillus oryzae, black thorn smoke pipe bacterium (Bjerkandera adusta), dry plan wax bacterium (Ceriporiopsis Aneirina), Ka Neiji intends wax bacterium (Ceriporiopsis caregiea), pale yellow plan wax pore fungi (Ceriporiopsis Gilvescens), the uncommon tower of Pernod intends wax bacterium (Ceriporiopsis pannocinta), annulus and intends wax bacterium (Ceriporiopsis Rivulosa), micro- red plan wax bacterium (Ceriporiopsis subrufa), worm intend wax bacterium (Ceriporiopsis Subvermispora), straight hem gold pityrosporion ovale (Chrysosporium inops), chrysosporium keratinophilum, Lu Kenuo trains of thought gold Pityrosporion ovale (Chrysosporium lucknowense), excrement shape gold pityrosporion ovale (Chrysosporium merdarium), rent Pityrosporion ovale, the golden pityrosporion ovale in Queensland, chrysosporium tropicum, brown thin golden pityrosporion ovale (Chrysosporium zonatum), tepetate ghost Umbrella (Coprinus cinereus), hairy fungus (Coriolus hirsutus), bar spore shape fusarium, cereal fusarium, storehouse prestige sickle Spore, machete fusarium, F.graminearum schw, red fusarium of standing grain, different spore fusarium, albizzia fusarium, sharp fusarium, racemosus fusarium, pink fusarium, connect Bone wood fusarium, colour of skin fusarium, intend branch spore fusarium, sulphur color fusarium, circle fusarium, plan silk spore fusarium, empiecement fusarium, special detritus Mould, Humicola lanuginosa, rice black wool mould, volume branch Mucor, thermophilic fungus destroyed wire, neurospora crassa, penicillium purpurogenum, yellow archespore Mao Pingge Bacterium, penetrate arteries and veins bacterium (Phlebia radiata), pleurotus eryngii (Pleurotus eryngii), autochthonal shuttle spore shell, domain Trametes trogii long (Trametes villosa), Trametes versicolor (Trametes versicolor), Trichoderma harzianum, trichodermaharzianum, long shoot trichoderma, Trichoderma reesei or Trichoderma viride cell.
Can be by fungal cell by being related to protoplast formation, protoplast transformation and the method for cell wall-deficient mutant Convert in a way known.For converting the suitable program of aspergillus and pyr-trichoderma host cell in the peace treaties of EP 238023 Er Dun (Yelton) et al., 1984, NAS's proceeding 81:1470-1474 and Harald Christensen (Christensen) et al., 1988, biology/technology (Bio/Technology) 6:Described in 1419-1422.For converting sickle The appropriate methodology of spore species is by horse traction Deere (Malardier) et al., 1989, gene (Gene) 78:147-156 and WO96/00787 is described.Can use by the program transformed yeast as described in documents below：Bake that (Becker) and melon human relations are special (Guarente), at Abbe Ademilson (Abelson), J.N. and simon (Simon), M.I. are compiled, yeast geneticses and molecular biosciences Learn guide, Enzymology method (Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology), volume 194, the 182-187 pages, Co., Ltd of academic press (Academic Press, Inc.), New York； Her rattan (Ito) et al., 1983, Bacteriology 153:163；And Hani grace (Hinnen) et al., 1978, National Science Institute's proceeding 75:1920.
Production method
Method the invention further relates to produce polypeptide, these methods include：A () trains under conditions of being suitable to express the polypeptide Support host cell of the invention；And (b) reclaims the polypeptide.In a preferred aspect, the cell is Aspergillus cell.At one Preferred aspect, the cell is Aspergillus oryzae cell.At a most preferred aspect, the cell is aspergillus oryzae MT3568.
These host cells are in the nutrient medium that method as known in the art produces the polypeptide is adapted for use with Culture.For example, can by Shaking culture, or in a kind of suitable culture medium and allow the expression of polypeptides and/or Small-scale or large scale fermentation is carried out in laboratory or industrial fermentation tank under conditions of separation (including to continuously ferment, send out in batches Ferment, batch feed fermentation or solid state fermentation) cultivate the cell.The culture is to use program as known in the art, in one kind It is adapted to generation in nutrient medium, the culture medium includes carbon and nitrogen source and inorganic salts.Suitable culture medium can be from commercial offers Business obtains or can be prepared according to disclosed composition (for example, in catalogue of American type culture collection).If many Peptide is secreted into the nutrient medium, then polypeptide directly can be reclaimed from culture medium.If polypeptide is not secreted, then it can Reclaimed from cell pyrolysis liquid.
Using specificity the polypeptide can be detected for the methods known in the art of these polypeptides.These detection methods Including but not limited to, the use of specific antibody, the formation of enzyme product or the disappearance of zymolyte.It is, for example possible to use enzymatic determination To determine the activity of the polypeptide.The example of lipase activity determination is as known in the art, including as described by instances Flat board determine and pNP determine.
Polypeptide can be reclaimed using methods known in the art.For example, the polypeptide can by conventional program, including but It is not limited to, collects, is centrifuged, filters, extracts, is spray-dried, evaporates or precipitates, is reclaimed from the nutrient medium.
The polypeptide can be purified by multiple programs as known in the art to obtain substantially pure polypeptide, these journeys Sequence is included but is not limited to：Chromatography is (for example, ion-exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing and chi Very little exclusion chromatography), electrophoretic procedures (for example, preparative isoelectric focusing), differential solubilities (for example, ammonium sulfate precipitation), SDS- PAGE is extracted and (be see, for example, protein purification (Protein Purification), Jansen (Janson) and bad step on (Ryden) Editor, VCH publishing houses (VCH Publishers), New York, 1989).
In a substituting aspect, the polypeptide is not recovered, but uses the host of the invention for expressing the polypeptide thin Born of the same parents originate as the polypeptide.
Composition
Also allow for including the composition of polypeptide of the invention.
In some aspects, the present invention relates to the polypeptide of the separation with lipase active, these separate polypeptides are selected from down Group, the group is made up of the following：(a) and SEQ ID NO:2 mature polypeptide have at least 60%, at least 65%, at least 70%th, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, The polypeptide of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity；B () is a kind of By the polypeptide of following polynucleotide encoding, the polynucleotides under low stringency condition, under middle stringent condition, in-high stringency conditions Under, under high stringency conditions or very under high stringency conditions with (i) SEQ ID NO:1 or SEQ ID NO:3 or (i) total length it is complementary Body hybridizes；A kind of (c) polypeptide, the polypeptide by with SEQ ID NO:1 or SEQ ID NO:3 have at least 60%, at least 65%, extremely Few 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%th, the polynucleotides of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity Coding；D a kind of () polypeptide, the polypeptide is to include substitution, missing at one or more (for example, a number) position, and/or insert The SEQ ID NO for entering:2 variant；And a kind of (e) polypeptide, the polypeptide is any one of the polypeptide of (a), (b), (c) or (d) Fragment.
In some aspects, the present invention relates to include the composition of polypeptide, the polypeptide is included in SEQ ID NO:2、SEQ ID NO:Substitution at one or more (for example, several) positions of 2 mature polypeptide or its fragment.In some respects, the present invention It is related to include the composition of polypeptide, the polypeptide is not changed in, i.e. be not included in SEQ ID NO:2、SEQ ID NO:2 Mature polypeptide or its fragment one or more (for example, several) positions at substitution.
The non-limiting list of the composition component being set forth below is suitable in these compositions, and method in this In some embodiments that can be eligibly incorporated herein, for example, it is used to aid in or strengthen clean-up performance, needs clearly for processing Clean substrate, or to modify the aesthetic feeling of said composition in the case of together with spices, colouring agent, dyestuff or the like.Mix The level for entering any such component in any combinations thing is in addition to the previously cited any material for incorporation.This The precise nature and its levels of incorporation of a little other components will depend on the physical form of composition and will wherein use combination The property of the clean operation of thing.Although being classified by general heading to component mentioned below according to specific feature, Be this and be not construed as limitation because as that will be understood by those of ordinary skill, a kind of component can include other function Property.
Unless otherwise indicated, amount in percentage is based on the weight of said composition (wt%).Suitable component material Including but not limited to surfactant, builder, chelating agent, dye transfer inhibitor, dispersant, enzyme and enzyme stabilizers, urge Change material, bleach-activating, hydrogen peroxide, hydrogen peroxide source, pre-formed peracid, polymeric dispersant, clay removal/resist again Deposition agent, brightener, foam in hibitors, dyestuff, dope dye, spices, perfume delivery system, the agent of structure elastic force, fabric-softening Agent, carrier, hydrotrote, processing aid, solvent and/or pigment.Except disclosed below, the suitable example of such other components with And use level is found in US 5576282, US 6306812 and US 6326348, is tied these documents hereby by quoting Close.
Therefore, in certain embodiments, one or more not comprising following attaching material of the present invention：Surfactant, Soap, builder, chelating agent, dye transfer inhibitor, dispersant, enzyme in addition, enzyme stabilizers, catalysis material, bleach-activating, Hydrogen peroxide, hydrogen peroxide source, pre-formed peracid, polymeric dispersant, clay removal/anti redeposition agent, brightener, foam It is inhibitor, dyestuff, spices, perfume delivery system, the agent of structure elastic force, fabric softener, carrier, hydrotrote, processing aid, molten Agent and/or pigment.However, in the presence of one or more component, such one or more component can be as detailed below What ground was present：
Surfactant- composition of the invention can include surfactant or surfactant system, wherein The surfactant can be selected from nonionic surface active agent, anion surfactant, cationic surfactant, both sexes Surfactant, zwitterionic surface-active agent, semi-polar nonionic surfactants and its mixture.When it is present, surface Activating agent typically with from 0.1wt% to 60wt%, from 0,2wt% to 40wt%, from 0,5wt% to 30wt%, from 1wt% to 50wt%, from 1wt% to 40wt%, from 1wt% to 30wt%, from 1wt% to 20wt%, from 3wt% to 10wt%, from 3wt% to 5wt%, from 5wt% to 40wt%, from 5wt% to 30wt%, from 5wt% to 15wt%, from 3wt% to 20wt%, Level from 3wt% to 10wt%, from 8wt% to 12wt%, from 10wt% to 12wt% or from 20wt% to 25wt% is present.
Suitable anionic detersive surfactant includes sulfate and sulfonate detersive surfactant.
Suitable sulfonate detersive surfactant includes alkylbenzenesulfonate, is on the one hand C10-13Alkyl benzene sulphonate Salt.Suitable alkylbenzenesulfonate (LAS), suitable LAB bags can be obtained by the commercially available linear alkylbenzene (LAB) of sulfonation (LAB) Low-carbon (LC) 2- phenyl LAB are included, such asOrOther suitable LAB include high-carbon 2- phenyl LAB, such asSuitable anionic detersive surfactant is the alkylbenzenesulfonate obtained by DETAL Catalytic processes, but Other route of synthesis (such as HF) can also be suitable.On the one hand, the magnesium salts of LAS is used.
Suitable sulphate detersive surfactant includes alkyl sulfate, is C on the one hand8-18Alkyl sulfate, or Predominantly C12Alkyl sulfate.
Other suitable sulphate detersive surfactant is alkyl alkoxylated suifate, is on the one hand alkyl second Epoxide sulfate, is on the one hand C8-18Alkyl alkoxylated suifate, is on the other hand C8-18Alkyl ethoxylated sulfuric acid Salt, typically alkyl alkoxylated suifate have from 0.5 to 20 or from 0.5 to 10 average degree of alkoxylation, typically alkane Alkoxylated sulphate is C8-18Alkyl ethoxylated sulfate, with 0.5 to 10, from 0.5 to 7, from 0.5 to 5 or from 0.5 To 3 average degree of ethoxylation.
Alkyl sulfate, alkyl alkoxylated suifate and alkylbenzenesulfonate can be straight or branched, substitution or It is unsubstituted.
Detersive surfactant can be the detersive surfactant of middle chain component, on the one hand for middle chain component it is cloudy from Sub- detersive surfactant, is on the one hand the alkyl sulfate and/or the alkylbenzenesulfonate of middle chain component of middle chain component, example Such as the alkyl sulfate of middle chain component.On the one hand, middle chain component is C1-4Alkyl, typically methyl and/or ethyl.
The non-limiting examples of anion surfactant include sulfate and sulfonate, specifically linear alkylbenzene (LAB) Sulfonate (LAS), the isomers of LAS, branch-alkylbenzene sulfonate (BABS), phenylalkane sulfonate, alpha-alkene sulfonate (AOS), alkene sulfonate, alkene sulfonates, alkane -2,3- diyls double (sulfate), hydroxy-alkanesulfonates and two sulphurs Hydrochlorate, alkyl sulfate (AS) (such as lauryl sodium sulfate (SDS)), aliphatic alcohol sulfate (FAS), primary alcohol sulfate (PAS), Ether alcohol sulfate (AES or AEOS or FES, also referred to as alcohol ethyoxysulfates or fatty alcohol ether sulphate), secondary alkanesulfonic acid Salt (SAS), paraffin sulfonate (PS), sulfonated ester, the fatty glyceride of sulfonation, α-sulfonic group fatty acid methyl ester (α- SFMe or SES) (including methyl ester sulfonate (MES)), alkyl succinic acid or alkenyl succinic acid, laurylene base/tetradecene base amber The diester and monoesters of acid (DTSA), the derivative of fatty acid of amino acid, sulfonic group butanedioic acid or soap, and combinations thereof.
Suitable non-ionic detersive surfactant is selected from the group, and the group is made up of the following：C8-C18Alkyl ethoxy Compound, such asC6-C12Alkyl phenol alkoxylates, wherein the alcoxylates unit can be ethyleneoxies Unit, propyleneoxy units or its mixture；C12-C18Alcohol and C6-C12Alkylphenol gathers with ethylene oxide/propylene oxide block The condensation product of compound, such as pluronicC14-C22The alcohol of middle chain component；C14-C22The alkyl of middle chain component Alcoxylates, typically with the average degree of alkoxylation from 1 to 30；Alkyl polysaccharide, is on the one hand APG；It is many Polyhydroxy fatty acid amides；Ether capped poly- (alkoxylate) alcohol surfactant；And its mixture.
Suitable non-ionic detersive surfactant includes APG and/or alkyl alkoxylated alcohol.
On the one hand, non-ionic detersive surfactant includes alkyl alkoxylated alcohol, is on the one hand C8-18Alkyl alkane Epoxide alcohol, such as C8-18Alkyl ethoxylated alcohol, the alkyl alkoxylated alcohol can have from 1 to 50, from 1 to 30, from 1 to 20 or from 1 to 10 average degree of alkoxylation.On the one hand, alkyl alkoxylated alcohol can be C8-18Alkyl ethoxylated alcohol, With from 1 to 10, from 1 to 7, it is more be from 1 to 5 or from 3 to 7 average degree of ethoxylation.Alkyl alkoxylated alcohol can be It is straight or branched and substituted or unsubstituted.Suitable nonionic surfactant includes
The non-limiting examples of nonionic surface active agent include alcohol ethoxylate (AE or AEO), alcohol propoxylation Thing, propenoxylated fatty alcohol (PFA), the fatty acid alkyl esters of alkoxylate are (such as ethoxylation and/or propenoxylated Fatty acid alkyl esters), alkylphenol ethoxylate (APE), nonyl phenol ethoxylate (NPE), APG (APG), alkane Epoxide amine, fatty monoethanol amide (FAM), fatty diglycollic amide (FADA), the aliphatic acid monoethanol of ethoxylation Acid amides (EFAM), propenoxylated fatty monoethanol amide (PFAM), polyhydroxy alkyl fatty acid acid amides, or gucosamine N- acyl N-alkyl derivatives (glucamide (GA), or fatty acid glucamides (FAGA)), together with SPAN and TWEEN commodity Obtainable product under one's name, and combinations thereof.
Suitable cationic detersive surfactants include alkyl pyridinium compounds, alkyl quaternary ammonium compound, alkyl quaternary Phosphonium compounds, the sulfonium compound of alkyl three and its mixture.
Suitable cationic detersive surfactants are the quaternary ammonium compounds with below general formula：(R)(R1)(R2)(R3)N+ X-, wherein R is straight or branched, substituted or unsubstituted C6-18Alkyl or alkenyl part, R1And R2Independently selected from methyl or Aminoethyl moiety, R3It is hydroxyl, methylol or hydroxyethyl moieties, X is to provide the anion of neutral charge, and suitable anion includes Halide, such as chloride；Sulfate；And sulfonate.Suitable cationic detersive surfactants are single-C6-18Alkyl List-hydroxyethyl dimethyl aliquat.The suitable cationic detersive surfactants of height are single-C8-10Alkyl list-hydroxyl second Base dimethyl quaternary ammonium chloride, single-C10-12Alkyl list-hydroxyethyl dimethyl aliquat and single-C10Alkyl list-hydroxyl second Base dimethyl quaternary ammonium chloride.
The non-limiting examples of cationic surfactant include alkyl dimethyl ethanol quaternary amine (ADMEAQ), cetyl Trimethylammonium bromide (CTAB), dimethyl distearyl ammonium chloride (DSDMAC) and alkyl benzyl dimethyl ammonium, quaternary ammonium alkyl Compound, alkoxy quaternary ammonium (AQA) compound, ester quaternary ammonium and combinations thereof.
Suitable amphoteric surfactant/zwitterionic surface-active agent includes amine oxide and glycine betaine (such as alkyl-dimethyl Base glycine betaine, sulfobetaines) or its combination.Anion surfactant-anion surface active that amine of the invention is neutralized Agent and attached anion cosurfactant can be existed by sour form, and the sour form can be neutralized to be formed It is desirably used for the surfactant salt of detergent composition of the present invention.The reagent for being typically used for neutralizing includes metal counter ion Alkali, such as hydroxide, such as NaOH or KOH.For neutralizing anion surfactant of the invention and in its sour form The other preferred reagent of attached anion surfactant or cosurfactant includes ammonia, amine or alkanolamine.It is preferred that alkanol Amine.Suitable non-limiting examples include MEA, diethanol amine, triethanolamine and other are as known in the art straight The alkanolamine of chain or side chain；For example, highly preferred alkanolamine includes 2- amino -1- propyl alcohol, 1- aminopropanols, single isopropanol Amine or 1- amino -3- propyl alcohol.In amine and the degree that may proceed to wholly or in part, for example, anion surfactant mixes The part of thing can be neutralized by sodium or potassium, and the part of anionic surfactant mixture can be by amine or alkanolamine With.
The non-limiting examples of Semi-polar surfactants include amine oxide (AO), such as alkyldimethylamine oxide
Comprising one or more anion surfactant and another or various nonionic surfactants and can Can be excellent optionally with the surfactant system of the mixture of other surfactant such as cationic surfactant Choosing.Preferred anion is at least 2 with the weight ratio of nonionic surfactant:1 or at least 1:1 to 1:10.
In certain embodiments of the present invention, said composition is selected from following surfactant or surfactant system： Neopelex, hydrogenated coconut oil sodium, sodium laureth sulfate, C12-14 alkanols polyethers -7, C12-15 alkanols polyethers - 7th, C12-15 alkanols polyethers sodium sulphate and C14-15 alkanols polyethers -4.
Soap-composition in this can include soap.Without being limited by theory, can it is desirable to include soap, because it Part serves as surfactant and builder is served as in part, and can be used to suppressing foam, and furthermore, it is possible to advantageously with Various cationic compounds of composition interact to strengthen the flexibility of the textile fabric processed with the present composition. Any soap for being used in laundry detergent compositions as known in the art can be utilized.In one embodiment, these combinations Thing includes the soap from 0wt% to 20wt%, from 0.5wt% to 20wt%, from 4wt% to 10wt% or from 4wt% to 7wt%.
The example of useful soap includes oleate soap, palmitic acid soap, palm kernel fatty acid soap and its mixture herein.Typical case Soap be in the form of the fatty acid soaps mixture with different chain length and substitution value.A kind of such mixture is topping palm Benevolence aliphatic acid.
In one embodiment, the soap is selected from free fatty.Suitable aliphatic acid be saturation and/or it is undersaturated and Can be from natural origin such as plant or animal ester (for example, palm-kernel oil, palm oil, coconut oil, babassu oil, safflower oil, Tuo Er Oil, castor oil, butter and fish oil, grease and its mixture) middle acquisition, or synthetically prepare (for example, via the oxidation of oil Or hydrogenate carbon monoxide via fischer tropsch process (Fisher Tropsch process)).
The example of the suitable saturated fatty acid for using in the compositions of the present invention includes capric acid, laurate, Pork and beans Cool acid, palmitic acid, stearic acid, arachidic acid are He behenic acid.Suitable unrighted acid species includes：Palmitoleic acid, oleic acid, Asia Oleic acid, leukotrienes and castor oil acid.The example of preferred aliphatic acid is saturation Cn aliphatic acid, saturation Ci2-Ci4Aliphatic acid and full And/or undersaturated Cn to Ci8Aliphatic acid and its mixture.
When it is present, fabric-softening cation cosurfactant is preferably from about 1 with the weight ratio of aliphatic acid:3 To about 3:1, more preferably from about 1:1.5 to about 1.5:1, most preferably about 1:1.
The level of soap in this and non-soap anionic surfactant be with acid basis specify by detergent composition Weight meter percentage.However, as being generally understood that in this area, in practice using sodium, potassium or alkanol ammonium alkali such as With anion surfactant and soap in NaOH or MEA.
Water-assisted solvent- composition of the invention can include one or more water-assisted solvent.Water-assisted solvent is following chemical combination Thing, the compound dissolves hydrophobic compound (or on the contrary, polar substances) in nonpolar environment in aqueous solution.Typically Ground, hydrotrote has hydrophilic and hydrophobic two kinds of features (such as by the amphipathic characteristic of surfactant known so-called)；However, helping The molecular structure of the hydrotrope is typically unfavorable for spontaneous self aggregation, see, for example, and is strangled by Huo Qideng (Hodgdon) and card (Kaler) (2007), colloid ＆ interface sciences are newly shown in (Current Opinion in Colloid＆Interface Science) 12：The summary of 121-128.Water-assisted solvent does not show critical concentration, will occur such as Surfactant higher than the concentration The found self aggregation of speech and lipid form micella, thin layer or other interphases for defining well.Many water-assisted solvents are anti- And continuous type accumulation process is shown, wherein the size of aggregation increases as concentration increases.However, many water-assisted solvents change The phase of the system of the material including polarity and apolar character (including mixture of water, oil, surfactant and polymer) Behavior, stability and colloid property.Classical ground is inter-trade water-soluble using helping to technology application from pharmacy, personal nursing, food Agent.The water-assisted solvent surfactant formulatory product for example denseer using permission in detergent compositions are (such as by removing Water and during compressed liquid detergent) without causing undesirable phenomenon, for example be separated or high viscosity.
Detergent can include from 0wt% to 10wt%, such as from 0wt% to 5wt%, 0.5wt% to 5wt% or from The water-assisted solvent of 3wt% to 5wt%.Can utilize and as known in the art any help water-soluble for what is used in detergent Agent.The non-limiting examples of water-assisted solvent include benzene sulfonic acid sodium salt, paratoluenesulfonic acid sodium salt (STS), sodium xylene sulfonate (SXS), withered Alkene sodium sulfonate (SCS), cymene sodium sulfonate, amine oxide, alcohol and polyglycol ether, hydroxynaphthoic acid sodium, croceine acid sodium, second Base hexyl sodium sulfonate and combinations thereof.
Builder- composition of the invention can include one or more builder, co-builder, builder system or its Mixture.When a builder is used, Cleasing compositions will typically comprise from 0wt% to 65wt%, at least 1wt%, from 2wt% Builder to 60wt% or from 5wt% to 10wt%.In tableware Cleasing compositions are washed, the level of builder is typically 40wt% to 65wt% or 50wt% to 65wt%.Said composition can be substantially free of builder；It is substantially free of the meaning " not intentionally adding " zeolite and/or phosphate.Typical zeolite builders include Wessalith CS, zeolite P and zeolite MAP.Allusion quotation The phosphate builder of type is sodium tripolyphosphate.
Builder and/or co-builder can be specifically the chelating for forming the water-soluble compound with Ca and Mg.Can To use any builder and/or co-builder for being used to be used in detergent as is generally known in the art.Builder it is unrestricted Property example include zeolite, diphosphate (pyrophosphate), triphosphate (such as sodium tripolyphosphate (STP or STPP)), carbonate (such as carbon Sour sodium), soluble silicate (such as sodium metasilicate), phyllosilicate (for example, from Hirst company (Hoechst) SKS- 6), monoethanolamine (such as 2- amino second -1- alcohol (MEA), imido grpup diethanol (DEA) and 2,2 ', 2 "-secondary Triaethanolamine (TEA)), And Carboxymethylinulin (CMI), with and combinations thereof.
Cleasing compositions can individually comprise co-builder, or and builder, such as zeolite builders combination.Help altogether and wash The non-limiting examples of agent include the homopolymers or its copolymer of polyacrylate, such as poly- (acrylic acid) (PAA) or copolymerization (third Olefin(e) acid/maleic acid) (PAA/PMA).Other non-limiting examples include citrate, chelating agent, such as aminocarboxylate, ammonia Hydroxypolycarboxylates and phosphonate, and alkyl-or alkenyl succinic acid.Other particular instance includes 2,2 ', 2 "-the second of secondary amino three Sour (NTA), ethylenediamine tetra-acetic acid (EDTA), diethylene-triamine pentaacetic acid (DTPA), imido disuccinic acid (IDS), ethylenediamine- N, N '-disuccinic acid (EDDS), MDGA (MGDA), glutamic acid-N, N- oxalic acid (GLDA), 1- hydroxyls ethane- 1,1- diyls double (phosphonic acids) (HEDP), ethylenediamine tetraacetic (methylene) four (phosphonic acids) (EDTMPA), (methylenes of diethylenetriamines five Base) five (phosphonic acids) (DTPMPA), N- (2- ethoxys) iminodiacetic acid (EDG), aspartic acid-N- list acetic acid (ASMA), day Winter propylhomoserin-N, N- oxalic acid (ASDA), aspartic acid-N- lists propionic acid (ASMP), imido disuccinic acid (IDA), N- (2- sulphur first Base) aspartic acid (SMAS), N- (2- sulfoethyls) aspartic acid (SEAS), N- (2- sulphurs methyl) glutamic acid (SMGL), N- (2- sulphurs Ethyl) glutamic acid (SEGL), N- methyliminodiacetic acids (MIDA), α-alanine-N, N- oxalic acid (α-ALDA), silk ammonia Acid-N, N- oxalic acid (SEDA), isoerine-N, N- oxalic acid (ISDA), phenylalanine-N, N- oxalic acid (PHDA), adjacent ammonia Yl benzoic acid-N, N- oxalic acid (ANDA), p-aminobenzene sulfonic acid-N, N- oxalic acid (SLDA), N-diacetic acid, N- oxalic acid And sulphur methyl-N, N- oxalic acid (SMDA), N- (ethoxy)-ethylene diamine triacetate (HEDTA), diethanol are sweet (TUDA) Propylhomoserin (DEG), diethylene triamine penta(methylene phosphonic acid) (DTPMP), amino three (methylene phosphonic acid) (ATMP), and combinations thereof And salt.Other exemplary builder and/or co-builder is described in such as WO 09/102854, US 5977053.
Chelating agent and crystal growth inhibitor- composition in this can include chelating agent and/or crystal growth inhibitor. Suitable molecule includes copper, ion and/or manganese chelating agent and its mixture.Suitable molecule includes DTPA (diethylenetriamines Pentaacetic acid), HEDP (hydroxyethane diphosphonic acid), DTPMP (diethylene triamine penta(methylene phosphonic acid)), 1,2- dihydroxy benzenes- 3,5- disulfonates salt hydroxide, ethylenediamine, diethylenetriamines, EDDS (EDDS), N- hydroxyethyls Ethylenediamine triacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N- hydroxyethyliminodiacetic acids (HEIDA), two Hydroxyethyl glycine (DHEG), the propionic acid of ethylene diamine four (EDTP), Carboxymethylinulin and 2- phosphono group butane 1,2,4- Tricarboxylic acids () and its derivative AM.Typically, said composition can include from 0.005wt% to 15wt% or from The chelating agent or crystal growth inhibitor of 3.0wt% to 10wt%.
Bleaching component- the bleaching component for being suitable for mixing in the method for the present invention and composition includes more than one bleaching One kind or mixture in component.Suitable bleaching component includes bleaching catalyst, optical white, bleach-activating, peroxidating Hydrogen, hydrogen peroxide source, pre-formed peracid and its mixture.Generally, when using bleaching component, composition of the invention can be with Including from 0wt% to 30wt%, from 0.00001wt% to 90wt%, 0.0001wt% to 50wt%, from 0.001wt% to 25wt% or from 1wt% to 20wt%.The example of suitable bleaching component includes：
(1) pre-formed peracid：Suitable pre-formed peracid includes but is not limited to the compound being selected from the group, the group by Preformed peroxy acid or its salt are constituted, typically peroxycarboxylic acid or its salt or peroxosulphuric or its salt.
Pre-formed peroxy acid or its salt are preferably peroxycarboxylic acid or its salt, typically with corresponding to below formula Chemical constitution：
Wherein：R14Selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic group；R14Group can be straight or branched , it is substituted or unsubstituted；And Y is any suitable counter ion for reaching neutral charge, preferably Y is selected from hydrogen, sodium or potassium. Preferably, R14It is straight chain or side chain, substituted or unsubstituted C6-9Alkyl.Preferably, peroxy acid or its salt are selected from peroxide Caproic acid, peroxide enanthic acid, Peroxycaprylic acid, pernoanoic acid, peroxydecanoic, its any salt or its any combinations.Particularly preferred peroxide Acid is phthalimide-based-peroxy-alkanoic acid, particularly ε-phthalimide-based peroxy caproic acid (PAP).It is excellent Selection of land, peroxy acid or its salt have the fusing point in the range of from 30 DEG C to 60 DEG C.
Pre-formed peroxy acid or its salt can also be peroxosulphuric or its salt, typically with corresponding to below formula Chemical constitution：
Wherein：R15Selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic group；R15Group can be straight or branched , it is substituted or unsubstituted；And Z is any suitable counter ion for reaching neutral charge, preferably Z is selected from hydrogen, sodium or potassium. Preferably, R15It is straight chain or side chain, substituted or unsubstituted C6-9Alkyl.Preferably, such bleaching component can by from 0.01wt% to 50wt% or the amount from 0.1wt% to 20wt% are present in composition of the invention.
(2) hydrogen peroxide source includes such as inorganic perhydrate salts, including alkali metal salt, such as perborate (generally It is monohydrate or tetrahydrate), percarbonate, persulfate, perphosphate, the sodium salt and its mixture of persilicate. One aspect of the present invention, inorganic perhydrate salts are those for being selected from the following group, and the group is made up of the following：Cross The sodium salt and its mixture of borate, percarbonate.When deployed, inorganic perhydrate salts are typically with entire combination thing 0.05wt% to 40wt% or the amount of 1wt% to 30wt% exist and be typically impregnated in into such composition as can With the crystalline solid being coated.Suitable coating includes：Inorganic salts, such as alkali silicate, carbonate or borate or its Mixture, or organic material, such as water-soluble or aqueous dispersion polymers, wax, oil or fat soap.Preferably, such bleaching group Dividing can be present in composition of the invention by the amount of 0.01wt% to 50wt% or 0.1wt% to 20wt%.
(3) term bleach-activating means with hydroperoxidation with via the chemical combination for crossing hydrolysis formation peracid herein Thing.The peracid for being formed in this way constitutes the bleaching agent of activation.Need suitable bleach-activating as used herein include belong to ester, Acid amides, acid imide or anhydrides it is other those.Suitable bleach-activating is those with R- (C=O)-L, and wherein R is alkane Base group (preferably side chain), when the bleach-activating is hydrophobic, with the carbon atom from 6 to 14 or from 8 To 12 carbon atoms, and when the bleach-activating is hydrophilic, having less than 6 carbon atoms or less than 4 carbon atoms；And And be L leaving groups.The example of suitable leaving group is the benzene sulfonate of benzoic acid and its derivative-especially.Suitable drift White activator includes lauroyl epoxide benzene sulfonate, decanoyloxybenzenesulphonate, decanoyloxybenzoic acid or its salt, 3,5,5- tri- Methyl hexanoyl epoxide benzene sulfonate, tetra acetyl ethylene diamine (TAED), 4- [(3,5,5- trimethyl acetyls base) epoxide] benzene -1- sulphurs Sour sodium (ISONOBS), 4- (dodecanoyl epoxide) benzene -1- sulfonate (LOBS), 4- (capryl epoxide) benzene -1- sulfonate, 4- (capryl epoxide) benzoate (DOBS or DOBA), 4- (pelargonyl group epoxide) benzene -1- sulfonate (NOBS)), and/or be disclosed in Those in WO 98/17767.The family of bleach-activating is disclosed in EP 624154 and particularly preferred in that family Be ATEC (ATC).ATC or short chain triglyceride (as triacetin) have advantages below, and it is environment Friendly.Additionally, ATEC and triacetin have good hydrolytic stability in storage in the product, and It is effective bleach-activating.Finally, ATC is multi-functional, because the citrate discharged in hydrolysis is crossed can be made For builder works.Alternately, bleaching system can include the peroxy acid of such as acid amides, acid imide or sulfone type.Bleaching system System can also include peracid, such as 6- (phthalimide-based) peroxy caproic acid (PAP).Suitable bleach-activating is also disclosed In WO98/17767.Although any suitable bleach-activating can be used, in one aspect of the invention, theme is clear Cleansing composition can include NOBS, TAED or its mixture.When it is present, based on fabric and household care composition, peracid and/ Or bleach-activating is generally present in the amount of 0.1wt% to 60wt%, 0.5wt% to 40wt% or 0.6wt% to 10wt% In composition.Can be by one or more hydrophobicity peracid or its precursor and one or more hydrophilic peracids or its combination of precursors Use.Preferably, such bleaching component can be present in this by the amount of 0.01wt% to 50wt% or 0.1wt% to 20wt% In the composition of invention.
The amount of hydrogen peroxide source and peracid or bleach-activating can be selected, to cause that available oxygen (comes from peroxide Compound source) with the mol ratio of peracid it is from 1:1 to 35:1, or even 2:1 to 10:1.
(4) diacyl peroxide-preferred diacyl peroxide bleaching species includes being selected from below general formula Those of diacyl peroxide：R1-C(O)-OO-(O)C-R2, wherein R1Represent C6-C18Alkyl, preferably comprising with extremely Lack 5 straight chains of carbon atom and optionally comprising one or more substitution bases (such as-N+(CH3)3,-COOH or-CN) and/or The C of the interrupt unit (such as-CONH- or-CH=CH-) that one or more are inserted between the adjacent carbon atom of alkyl6-C12Alkane Base group, and R2Represent and the compatible aliphatic group of peroxide portion, to cause R1And R2Together comprising 8 altogether To 30 carbon atoms.In a preferred aspect, R1And R2It is the unsubstituted C of straight chain6-C12Alkyl chain.Most preferably, R1And R2 It is identical.Diacyl peroxide (wherein R1And R2It is C6-C12Alkyl group) it is particularly preferred.Preferably, R group (R1Or R2) at least one of, most preferably only have one and do not include at α branch or sagging ring, or preferably at α or β all not comprising branch or sagging ring, or most preferably in α or β or γ all not comprising branch or sagging ring. In a further preferred embodiment, DAP can be asymmetric, to cause that the hydrolysis of preferably R1 carboxyl groups is fast Speed to produce peracid, but the hydrolysis of R2 carboxyl groups is slow.
Four acyl peroxides bleaching species is preferably selected from four acyl peroxides with below general formula：R3-C(O)- OO-C(O)-(CH2)n-C(O)-OO-C(O)-R3, wherein R3Represent C1-C9Alkyl, or C3-C7Group, and n represented from 2 to 12 Or the integer of 4 to 10 (including end value).
Preferably, diacyl and/or four acyl peroxides bleaching species with enough offer at least 0.5ppm by weight, The amount of the cleaning solution of at least 10ppm or at least 50ppm is present.In a preferred embodiment, these bleach species to carry enough Amount for cleaning solution by weight from 0.5ppm to 300ppm, from 30ppm to 150ppm is present.
Preferably, bleaching component includes bleaching catalyst (5 and 6).
(5) preferably organic (nonmetallic) bleaching catalyst, including the oxygen from peroxy acid and/or its salt can be received Atom and by the bleaching catalyst of the Oxygen atom transfer to oxidable substrate.Suitable bleaching catalyst includes but does not limit In：Iminium cations and polyion；Imines hybrid ion；Modified amine；Modified oxidized amine；N- sulphonyl imines；N- phosphonos Base imines；N- acyl imines；Thiadiazoles dioxide；Perfluor imines；Cyclic sugar and its mixture.
Suitable iminium cations and polyion are included but is not limited to, N- methyl -3,4- dihydro-isoquinoline tetrafluoro boron Hydrochlorate, such as tetrahedron (Tetrahedron) (1992), 49 (2), being prepared (for example, compound 4, described in 423-38 Page 433)；N- methyl -3, the p- toluene fulfonate of 4- dihydro-isoquinolines is prepared (such as as described in US 5360569 11 columns, example 1)；And n-octyl -3, the p- toluene fulfonate of 4- dihydro-isoquinolines, the carrying out as described in US 5360568 make Standby (such as the 10th column, example 3).
Suitable imines hybrid ion is included but is not limited to, N- (3- sulfopropyls) -3,4- dihydro-isoquinolines, inner salt, Being prepared on (such as the 31st column, example II) as described in US 5576282；N- [2- (sulphur epoxide) dodecyl] -3,4- dihydros Isoquinolin, inner salt, being prepared on (for example, the 32nd column, example V) as described in US 5817614；2- [3- [(2- ethyl hexyls Base) epoxide] -2- (sulphur epoxide) propyl group] -3,4- dihydro-isoquinolines, inner salt, being prepared as described in WO 05/047264 (for example, page 18, example 8), and 2- [3- [(2- butyl octyls) epoxide] -2- (sulphur epoxide) propyl group] -3,4- dihydro isoquinolines Quinoline, inner salt.
Suitable modified amine oxygen transfer catalyst includes but is not limited to 1,2,3,4- tetrahydrochysene -2- methyl isophthalic acids-isoquinolin alcohol, its Can be according to being described in Tet Lett (Tetrahedron Letters) (1987), 28 (48), the method system in 6061-6064 It is standby.Suitable modified oxidized amine oxygen transferring catalyst include but is not limited to 1- hydroxy-ns-epoxide-N- [2- (sulphur epoxide) decyl]- 1,2,3,4- tetrahydroisoquinoline sodium.
Suitable N- sulphonyl imine oxygen transferring catalysts are included but is not limited to according to Journal of Organic Chemistry (Journal of Organic Chemistry) (1990), 55 (4), 3- methyl isophthalic acids prepared by the program described in 1254-61,2- benzisothiazoles 1,1- dioxide.
Suitable N- phosphonyl imine oxygen transfer catalysts include but is not limited to [R- (E)]-N- [(chloro- 5- nitrobenzene of 2- Base) methylene]-to phenyl-p- (2,4,6- trimethylphenyl) phosphinic acid amide, its can according to being described in Chemical Society's magazine, Chemical communication (Journal of the Chemical Society, Chemical Communications) (1994), (22), It is prepared by the method in 2569-70.
Suitable N- acyl imine oxygen transferring catalyst including but not limited to can be according to Polish The Chemicals (Polish Journal of Chemistry) (2003), 77 (5), [N (E)]-N- (phenyl methylidenes of the program manufacture described in 577-590 Base) acetamide.
Suitable thiadiazoles dioxide oxygen transferring catalyst including but not limited to can according to US5753599 (the 9th column, Example 2) described in program manufacture 3- methyl 4-phenyl -1,2,5- thiadiazoles 1,1- dioxide.
Suitable perfluor imines oxygen transfer catalyst includes but is not limited to fluoro- N- (the nonyl fluorine of (Z) -2,2,3,3,4,4,4- seven Butyl) butyryl imines fluoride, its can according to Tet Lett (Tetrahedron Letters) (1994), 35 (34), It is prepared by the method described in 6329-30.
Suitable cyclic sugar oxygen transferring catalyst is included but is not limited to such as in US 6649085 (the 12nd column, example 1) 1,2 for preparing:Red -2,3- acetyl butyryls (the hexodiuro) -2,6- pyranoses of-O- isopropylidenes-D- of 4,5- bis-.
Preferably, the bleaching catalyst includes imines and/or carbonyl functional group, and usually can receive oxygen original Son, especially from peroxy acid and/or its salt receive oxygen atom after formed phenoxy imine cation (oxaziridinium) and/or Dioxirane functional group.Preferably, bleaching catalyst includes phenoxy imine cation functional group and/or is receiving oxygen atom When, especially can form phenoxy imine cation functional group when the oxygen atom from peroxy acid and/or its salt is received.It is preferred that Ground, bleaching catalyst include cyclic imide functional group, preferably wherein the annulus have from five to eight atom (including Nitrogen-atoms), preferably six ring sizes of atom.Preferably, bleaching catalyst includes arylimine functional groups, preferably two cyclophanes Base imine, preferably 3,4- dihydro-isoquinolines functional group.Typically, imine be season imine and Typically when oxygen atom is received, especially season peroxide can be formed when the oxygen atom from peroxy acid and/or its salt is received Cationic imide functional group.On the other hand, the detergent composition include have no more than 0, be not more than -0.5, be not more than - 1.0th, it is not more than -1.5, is not more than -2.0, logP for being not more than -2.5, being not more than -3.0 or be not more than -3.5o/wBleaching group Point.Describe in more detail below for determining logPo/wMethod.
Typically, bleach can be produced with from 0.01 to 0.30, from 0.05 to 0.25 or from 0.10 to 0.20 XSOBleaching species.Describe in more detail below for determining XSOMethod.For example, the bleaching with isoquinoline structure Composition can produce the bleaching species with phenoxy imine cation structure.In this example, XSOIt is phenoxy imine cation Bleach the X of speciesSO。
Preferably, bleaching catalyst has the chemical constitution corresponding to below formula：
Wherein：N and m are independently that preferably n and m is 0 from 0 to 4；Each R1Independently selected from substitution or unsubstituted The group being selected from the group, the group is made up of the following：Hydrogen, alkyl, cycloalkyl, aryl, fused-aryl, heterocycle, condense it is miscellaneous Ring, nitro, halogen, cyano group, sulfonate radical, alkoxy, ketone group, carboxyl and alkoxy carbonyl group；And the R of any two vicinal1Take Can merge to form fused-aryl, fused iso or annelated heterocycles for base；Each R2Independently selected from substitution or unsubstituted The group independently selected from the following group, the group is made up of the following：Hydrogen, hydroxyl, alkyl, cycloalkyl, alkaryl, aryl, virtue Alkyl, alkylidene, heterocycle, alkoxy, aryl carbonyl, carboxyalkyl and amide group；Any R2Can be with any other R2 It is combined together to form a part for common ring；It is any together with R2Can merge to form carbonyl；And any two R2Can close And to form the substituted or unsubstituted unsaturated part for condensing；R3It is C1To C20Substituted or unsubstituted alkyl；R4It is hydrogen Or Qt- part A, wherein：Q is that the alkene of branch or non-branch, t=0 or 1, and A are the anionic groups being selected from the group, should Group is made up of the following：OSO3 -、SO3 -、CO2 -、OCO2 -、OPO3 2-、OPO3H-And OPO2 -；R5It is hydrogen or-CR11R12-Y-Gb- Yc-[(CR9R10)y-O]k-R8Part, wherein：Independently selected from the following group, the group is made up of each Y the following：O, S, N-H or N-R8；And each R8Independently selected from the following group, the group is made up of the following：Alkyl, aryl and heteroaryl, the part is It is substituted or unsubstituted, and either replace or it is unsubstituted, the part is having less than 21 carbon；Each G independence Be selected from the group, the group is made up of the following：CO、SO2, SO, PO and PO2；R9And R10Independently selected from the following group, the group by with Lower every composition：H and C1-C4Alkyl；R11And R12Independently selected from the following group, the group is made up of the following：H and alkyl, or Carbonyl is may be combined to form when putting together；B=0 or 1；C can with=0 or 1, but if b=0, c it is necessary=0；Y be from 1 to 6 integer；K is the integer from 0 to 20；R6It is H, or alkyl, aryl or heteroaryl moieties；The part is substitution or does not take Generation；And X, if it exists, being suitable charge balancing counter ion, preferably works as R4When being hydrogen, X is exist, and is fitted The X of conjunction is included but is not limited to：Chloride, bromide, sulfate, methoxy sulfate (methosulphate), sulfonate, to first Benzene sulfonate, boron tetrafluoride and phosphate.
In one embodiment of the invention, bleaching catalyst has the structure corresponding to below general formula：
Wherein R13It is to include from three to 24 the branched alkyl of carbon atom (including carbon atom of branch) or comprising from one Individual to 24 straight chained alkyls of carbon atom；Preferably, R13Be include from eight to 18 the branched alkyl of carbon atom or comprising from Eight to 18 straight chained alkyls of carbon atom；Preferably, R13It is selected from the group, the group is made up of the following：2- propylheptyls, 2- butyl octyls, 2- pentylnonanyis, 2- hexyls decyl, n- dodecyl, n- myristyl, n- cetyl, n- 18 Alkyl, iso- nonyl, iso- decyl, iso- tritriacontyl and iso- pentadecyl；Preferably, R13It is selected from the group, the group is by the following Composition：2- butyl octyls, 2- pentylnonanyis, 2- hexyls decyl, iso- tritriacontyl and iso- pentadecyl.
Preferably, in addition to bleaching catalyst, particularly organic bleaching catalyst, bleaching component also includes source of peracid. Source of peracid can be selected from (a) pre-formed peracid；(b) percarbonate, perborate or persulfate (hydrogen peroxide source), preferably Combined with a kind of bleach-activating；(c) Perhydrolase and ester, in textile or crust process step in water In the presence of be formed in situ peracid.
When it is present, based on said composition, peracid and/or bleach-activating generally with from 0.1wt% to 60wt%, from 0.5wt% to 40wt% or the amount from 0.6wt% to 10wt% are present in said composition.Can be by one or more hydrophobicity Peracid or its precursor are used with one or more hydrophilic peracids or its combination of precursors.
The amount of hydrogen peroxide source and peracid or bleach-activating can be selected, to cause that available oxygen (comes from peroxide Compound source) with the mol ratio of peracid it is from 1:1 to 35:1, or 2:1 to 10:1.
(6) bleaching catalyst comprising metal-bleaching component can be provided by catalytic metal complex.A type of bag Bleaching catalyst containing metal is following catalysis system, and the catalysis system includes a kind of mistake with the bleach catalyst for limiting activity Metal cation (such as copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cation) is crossed, one kind has seldom or lived without bleach catalyst The auxiliary metal cation (such as zinc or aluminium cations) of property, and one kind has for catalytic and complementary metal cation The spacer of the stability constant of restriction, particularly ethylenediamine tetra-acetic acid, EDTMP and its water soluble salt. Such catalyst is disclosed in US 4430243.Preferred catalyst is described in WO 09/839406, US 6218351 and WO In 00/012667.Particularly preferably transition-metal catalyst or therefore as the multiple tooth N- donors of bridge (cross-bridged) The part of part.
If desired, composition in this can be catalyzed by manganese compound.Such compound and use level It is in the art the catalyst based on manganese well known and including being for example disclosed in US 5576282.
Cobalt bleaching catalyst useful herein is known and is for example described in US 5597936；In US 5595967. Such Co catalysts, the journey taught in such as US 5597936 and US 5595967 can easily be prepared by known program Sequence.
Composition in this can also compatibly include the transition metal complex of part, such as bipiperidine ketone (bispidone) (US 7501389) and/or macropolycyclic rigid ligand-be abbreviated as " MRL ".As a practical problem not It is used as limitation, composition in this and method can be adjusted, provides about at least about 100,000,000 points in Aqueous wash medium One of active MRL species, and will typically be in providing in cleaning solution from 0.005ppm to 25ppm, from 0.05ppm to The 10ppm or MRL from 0.1ppm to 5ppm.
Transition metal bleach catalyst in suitable transition metal include such as manganese, iron and chromium.Suitable MRL Including 5,12- diethyl -1,5,8,12- 4-azabicyclo [6.6.2] hexadecane.Can easily be prepared by known program suitable The program taught in the transition metal M RL of conjunction, such as US 6225464 and WO 00/32601.
(7) optical white-suitable optical white includes Phthalocyanine Zinc, the aluminum phthalocyanine of sulfonation, the xanthene dye of such as sulfonation And its mixture.Preferred bleaching component for being used in these compositions of the invention includes that hydrogen peroxide source, bleaching are lived Agent and/or organic peroxide acid, optionally by hydrogen peroxide source and bleach-activating and the combined reaction of bleaching catalyst And generation in situ.Preferred bleaching component includes bleaching catalyst, preferably above-described organic bleaching catalyst.
Particularly preferred bleaching component is bleaching catalyst, particularly organic bleaching catalyst.
Exemplary bleaching system is also described in such as WO 2007/087258, WO 2007/087244, WO 2007/ In 087259 and WO 2007/087242.
Fabric hueing agent- said composition can include fabric hueing agent.Suitable fabric hueing agent include dyestuff, dyestuff- Clay conjugates and pigment.Suitable dyestuff includes small molecule dyes and polymeric dye.Suitable small molecule dyes include The small molecule dyes being selected from the group, the group is made up of the following：Belong to the dyestuff of following color index (C.I.) classification：Directly Blue, direct red, direct purple, acid blue, acid red, acid violet, alkali blue, alkalescence purple and alkaline red or its mixture.
On the other hand, suitable small molecule dyes include the small molecule dyes being selected from the group, and the group is by the following group Into：Color index (dyer and colourist association (Society of Dyers and Colorists), Bradford, Britain) Numbering directly purple 9, directly purple 35, directly purple 48, directly purple 51, directly purple 66, directly purple 99, directly blue 1, directly blue 71, it is straight Connect blue 80, directly indigo plant 279, azogeramine 7, acid red 73, acid red 88, azogeramine 50, acid violet 15, acid violet 17, acidity Purple 24, acid violet 43, acid red 52, acid violet 49, acid violet 50, Blue VRS 5, Blue VRS 7, acid blue 25, acid blue 29, Acid Blue 40, acid blue 45, Acid Blue 75, acid blue 80, acid blue 83, acid blue 90 and Acid blue 113, acid black 1, alkalescence Purple 1, alkaline purple 3, alkalescence purple 4, alkaline purple 10, alkaline purple 35, alkali blue 3, alkali blue 16, alkali blue 22, alkali blue 47, alkalescence Blue 66, Blue 75, alkali blue 159 and its mixture.On the other hand, suitable small molecule dyes are small including what is be selected from the group Molecular dye, the group is made up of the following：Color index (dyer and colourist learn, Bradford, Britain) numbering acid Property purple 17, acid violet 43, acid red 52, acid red 73, acid red 88, azogeramine 50, acid blue 25, acid blue 29, acid blue 45th, Acid blue 113, directly acid black 1, blue 1, direct indigo plant 71, direct purple 51 and its mixture.On the other hand, it is suitable small Molecular dye includes the small molecule dyes being selected from the group, and the group is made up of the following：(dyer and colourist learn color index Meeting, Bradford, Britain) numbering acid violet 17, directly blue 71, directly purple 51, directly blue 1, acid red 88, azogeramine 50, Acid blue 29, Acid blue 113 or its mixture.
Suitable polymeric dye includes the polymeric dye being selected from the group, and the group is made up of the following：Comprising conjugated The polymer (dye-polymer conjugates) of chromogen and the polymer entered in main polymer chain with chromogen combined polymerization, and Its mixture.
On the other hand, suitable polymeric dye includes the polymeric dye being selected from the group, and the group is by the following group Into：Fabric substance colouring agent under (Milliken (Milliken)) title, from least one reactivity The dye-polymer conjugates that dyestuff and the polymer being selected from the group are formed, the group is made up of the following：Comprising selected from by hydroxyl The polymer of the part of the group of base section, one-level amine moiety, secondary amine part, thiol moiety and its mixture composition.Another again On the one hand, suitable polymeric dye includes the polymeric dye being selected from the group, and the group is made up of the following： Purple CT, and reactive blue, reactive violet or the conjugated carboxymethylcellulose calcium of active red dye (CMC), such as with C.I. reactive blues 19 (by wheat lattice enzyme (Megazyme), Wicklow, Ireland, with ProductName AZO-CM-CELLULOSE, is sold under product code S-ACMC Sell) conjugated CMC, the triphenyl-methane polymeric colorant of alkoxylate, the thiophene polymeric colorant of alkoxylate and Its mixture.
Preferred dope dye includes findings that the brightening agent in WO 08/87497.These brightening agents can be by following knot Structure (I) is characterized：
Wherein R1And R2Can be independently selected from：
a)[(CH2CR'HO)x(CH2CR"HO)yH]
Wherein R ' is selected from the group, and the group is made up of the following：H、CH3、CH2O(CH2CH2O)zH and its mixture；Wherein R " is selected from the group, and the group is made up of the following：H、CH2O(CH2CH2O)zH and its mixture；Wherein x+y≤5；Wherein y >= 1；And wherein z=0 to 5；
b)R1=alkyl, aryl or aryl alkyl, and R2=[(CH2CR'HO)x(CH2CR"HO)yH]
Wherein R ' is selected from the group, and the group is made up of the following：H、CH3、CH2O(CH2CH2O)zH and its mixture；Wherein R " is selected from the group, and the group is made up of the following：H、CH2O(CH2CH2O)zH and its mixture；Wherein x+y≤10；Wherein y >= 1；And wherein z=0 to 5；
c)R1=[CH2CH2(OR3)CH2OR4] and R2=[CH2CH2(OR3)CH2OR4]
Wherein R3It is selected from the group, the group is made up of the following：H、(CH2CH2O)zH and its mixture；And wherein z= 0 to 10；
Wherein R4It is selected from the group, the group is made up of the following：(C1-C16) alkyl, aromatic yl group and its mixture；And And
(d) wherein R1 and R2 can independently selected from styrene oxide, glycidol methyl ether, isobutyl glycidyl ether, Isopropyl glycidyl ether, tertiary butyl glycidyl ether, 2- hexyl glycidyl ethers and glycidol cetyl ether Amino addition compound product, be then from 1 to 10 addition of alkylene oxide unit.
Preferred whitening agent of the invention can be characterized by following structure (II)：
Wherein R ' is selected from the group, and the group is made up of the following：H、CH3、CH2O(CH2CH2O)zH and its mixture；Wherein R " is selected from the group, and the group is made up of the following：H、CH2O(CH2CH2O)zH and its mixture；Wherein x+y≤5；Wherein y >= 1；And wherein z=0 to 5.
Brightening agent preferred in addition of the invention can be characterized by following structure (III)：
Typically comprise with 5 mixtures of EO groups altogether.Suitable preferred molecule be in structure I with Under side base above in " part a " those.
Table A
R1 R2
R’ R” x y R’ R” x y
a H H 3 1 H H 0 1
b H H 2 1 H H 1 1
C=b H H 1 1 H H 2 1
D=a H H 0 1 H H 3 1
The other brightening agent for using includes being described in that in US 2008/34511 (Uniliver (Unilever)) A bit.Preferred reagent is " purple 13 ".
Suitable dye clay conjugates includes the dye clay conjugates that is selected from the group, the group include at least one sun from Son/basic-dyeable fibre and terre verte and its mixture.On the other hand, suitable dye clay conjugates includes the dye being selected from the group Material clay conjugates, the group is made up of a kind of cation/basic-dyeable fibre and a kind of clay, and the cation/basic-dyeable fibre is selected from down Group, the group is made up of the following：C.I. basic yellow 1 to 108, C.I. Basic Oranges 1 to 69, C.I. alkali red 1:1s to 118, C.I. Alkaline purple 1 to 51, C.I. alkali blues 1 to 164, C.I. Viride Nitenses 1 to 14, C.I. alkalescence brown 1 to 23, CI basic blacks 1 to 11, And the clay is selected from the group, the group is made up of the following：Montmorillonitic clay, hectorite clay, saponite clay and its mixing Thing.At yet another aspect, suitable dye clay conjugates includes the dye clay conjugates being selected from the group, and the group is by following Item composition：Montmorillonite alkali blue B7C.I.42595 conjugates, montmorillonite alkali blue B9C.I.52015 conjugates, montmorillonite alkalescence Red R1C.I.45160 is conjugated for purple V3C.I.42555 conjugates, montmorillonite Viride Nitens G1C.I.42040 conjugates, montmorillonite alkalescence Thing, the conjugates of montmorillonite C.I. basic blacks 2, hectorite alkali blue B7C.I.42595 conjugates, hectorite alkali blue B9C.I.52015 conjugates, hectorite alkalescence purple V3C.I.42555 conjugates, G1C.I.42040 is conjugated for hectorite Viride Nitens Thing, the red R1C.I.45160 conjugates of hectorite alkalescence, the conjugates of hectorite C.I. basic blacks 2, saponite alkali blue B7C.I.42595 conjugates, saponite alkali blue B9C.I.52015 conjugates, saponite alkalescence purple V3C.I.42555 conjugates, soap Stone Viride Nitens G1C.I.42040 conjugates, the red R1C.I.45160 conjugates of saponite alkalescence, the conjugates of saponite C.I. basic blacks 2 and Its mixture.
Suitable pigment includes the pigment being selected from the group, and the group is made up of the following：Yellow scholar's ketone, indanthrone, comprising 1 to 4 the chloride indanthrone of chlorine atom, pyranthrone, dichloro pyranthrone, single bromine dichloro pyranthrone, Dibromo-dichloro pyranthrone, tetrabromo skins (wherein the imide group can be unsubstituted or by C1- for anthrone, perylene -3,4,9,10- tetracarboxylic acid diimides C3- alkyl or phenyls or heterocyclic group substitution, and wherein the phenyl and heterocyclic group can additionally with not assigning Deliquescent substitution base in water), anthrapyrimidine carboxylic acid amide, violanthrone, isoviolanthrone, triazine dioxin pigments, each molecule can With chloro- comprising many bromines of up to 2 CuPcs of chlorine atom, how chloro- CuPc or each molecule comprising up to 14 bromine atoms CuPc, and its mixture.
On the other hand, suitable pigment includes the pigment being selected from the group, and the group is made up of the following：Ultramarine (C.I. Alizarol saphirol 29), ultramarine violet (C.I. pigment violet 1s 5) and its mixture.
Above-mentioned fabrics toner can be applied in combination (any mixture that can use fabric hueing agent).Suitable toning Agent is described in greater detail in US 7208459.Dyestuff preferred levels in the present compositions be 0.00001wt% extremely 0.5wt% or 0.0001wt% to 0.25wt%.It is preferred that be used for the concentration of the dyestuff for the treatment of and/or cleaning in water being From 1ppb to 5ppm, 10ppb to 5ppm or 20ppb to 5ppm.In preferred composition, the concentration of surfactant will be from 0.2 to 3g/l.
Capsule compound- said composition can include capsule compound.On the one hand, capsule compound includes a core, has Inner surface and the involucrum of outer surface, the encapsulated core of involucrum.
In the one side of the capsule compound, the core can include a kind of material being selected from the group, the group by with Lower every composition：Spices；Brightener；Dyestuff；Pest repellant；Silicone；Wax；Flavor enhancement；Vitamin；Fabric softener；Skin conditioner, It is on one side paraffin；Enzyme；Antiseptic；Bleaching agent；Feelings agent (sensate)；And its mixture；And the involucrum can be wrapped A kind of material being selected from the group is included, the group is made up of the following：Polyethylene；Polyamide；Polyvinyl alcohol, optionally comprising it His comonomer；Polystyrene；Polyisoprene；Makrolon；Polyester；Polyacrylate；Aminoplast, in one aspect, The aminoplast can include polyureas, polyurethane and/or polyurea polyurethanes (polyureaurethane), in one aspect, The polyureas can include polyoxy methylene urea and/or melamino-formaldehyde；Polyolefin；Polysaccharide is in one aspect, described more Sugar can include alginate and/or shitosan；Gelatin；Shellac；Epoxy resin；Polyvinyl water insoluble inorganic substance； Silicone；And its mixture.
In the one side of the capsule compound, the core can include spices.
In the one side of the capsule compound, the involucrum can include the trimerization of melamino-formaldehyde and/or crosslinking Cyanamide formaldehyde.
On the one hand, disclosing suitable capsule compound can include core material and involucrum, and the involucrum is at least part of Ground surrounds the core material.The 85% of the capsule compound or 90% can have from 0.2MPa to 10MPa, from 0.4MPa to 5MPa, the breaking strength from 0.6MPa to 3.5MPa or from 0.7MPa to 3MPa；And with from 0 to 30%, from 0 to 20%, Or from 0 to 5% beneficial agent reveal.
On the one hand, 85% or the 90% of the capsule compound can have from 1 to 80 micron, from 5 to 60 microns, from 10 To 50 microns or from 15 to 40 microns of granularity.
On the one hand, 85% or the 90% of the capsule compound can have from 30 to 250nm, from 80 to 180nm or from The particle wall thickness of 100 to 160nm.
On the one hand, the core material of the capsule compound can include a kind of selected from the group being made up of spices raw material Material, and/or optionally include a kind of material being selected from the group, the group is made up of the following：Vegetable oil, including pure plant Oil and/or blending vegetable oil, including castor oil (caster oil), coconut oil, cottonseed oil, grape-kernel oil, rapeseed, soybean Oil, corn oil, palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconut oil, palm-kernel oil, castor oil (castor Oil), lemon oil and its mixture；The ester of vegetable oil, ester, including dibutyl adipate, Dibutyl phthalate, adipic acid butyl benzyl Ester, adipic acid octyl group benzyl ester, tricresyl phosphate, trioctyl phosphate and its mixture；Straight or branched hydrocarbon, including with being higher than Those straight or branched hydrocarbon of about 80 DEG C of boiling point；Partially hydrogenated terphenyl, dialkyl phthalate, alkyl biphenyl (including single isopropyl biphenyl), the naphthalene (including dipropyl naphthalene) of alkylation, benzin (including kerosene), mineral oil and its mixing Thing；Aromatic solvent, including benzene, toluene and its mixture；Silicone oil；And its mixture.
On the one hand, the wall material of the capsule compound can include suitable resin, and the resin includes the anti-of aldehyde and amine Answering product, suitable aldehyde includes formaldehyde.Suitable amine includes melamine, urea, benzoguanamine, glycoluril and its mixture.It is adapted to Melamine include melamine methylol, the melamine methylol, imino group melamine and its mixture that methylate. Dimethylol urea, urea-resorcinol and its mixture that suitable urea includes dimethylol urea, methylates.
On the one hand, by capsule compound, added to before, during or after composition, suitable formaldehyde scavenger can be with Be used together for instance in the capsule compound in capsule slurry and/or added in this composition.Suitable capsule can be with By US 2008/0305982；And/or the following of US 2009/0247449 is taught to be made.
In a preferred aspect, said composition can also include deposition acid, be preferably made up of the following group, and the group includes cation Or non-ionic polymers.Suitable polymer includes cationic starch, cationic hydroxy ethyl cellulose, polyvinyl formal, Chinese scholartree Tree bean gum, mannosan, xyloglucan, tamarind gum, polyethylene terephthalate, and comprising dimethylaminoethyl methyl-prop Olefin(e) acid ester and the optional monomer that there is one or more to be selected from the group polymer, the group includes acrylic acid and acrylamide.
Spices- on the one hand, said composition is included containing one or more spices of spices raw material being selected from the group, should Group is made up of the following：Double -2- the propyl alcohol of 1,1'- epoxides；Isosorbide-5-Nitrae-cyclohexane dicarboxylic acid, diethyl ester；((ethoxymethyl) epoxide) Cyclododecane；1,3- nonanediol, monoacetate；(3- methylbutoxy groups) acetic acid, 2- propylene base esters；Beta-methyl cyclododecane second Alcohol；2- methyl -3- [(ring [2.2.1] the hept- 2- of 1,7,7- trimethyls two yls) epoxide] -1- propyl alcohol；- 2- the ketone of oxa- ring 16；α- Methyl-phenmethylol acetate；Trans -3- ethyoxyls -1,1,5- trimethyl-cyclohexanes；4- (1,1- dimethyl ethyls) cyclohexanol second Acid esters；Ten dihydro -3a, 6,6,9a- tetramethyl naphtho- [2,1-b] furans；Beta-methyl benzenpropanal；Beta-methyl -3- (1- methyl second Base) benzenpropanal；4- Phenyl 2 butanones；2-Methyl Butyric Acid, ethyl ester；Benzaldehyde；2-Methyl Butyric Acid, 1- Methylethyl esters；Two Hydrogen -5- amyl groups -2 (3H) furanone；(2E)-1- (2,6,6- trimethyl-2- cyclohexene-1- bases)-2- butene-1 -one；Lauric aldehyde； The hendecanal；2- ethyls-alpha, alpha-dimethyl benzenpropanal；Capraldehyde；Alpha, alpha-dimethyl benzyl carbinol acetic acid esters；2- (phenylmethylene) is pungent Aldehyde；2- [[3- [4- (1,1- dimethyl ethyl) phenyl] -2- methyl propylenes] amino] benzoic acid, methyl ester；1- (2,6,6- tri- Methyl-3- cyclohexene-1- bases)-2- butene-1 -one；2- amyl group cyclopentanone；3- oxo -2- pentyl-cyclopentane acetic acid, methyl ester； 3-methoxy-4-hydroxybenzaldehyde；Vanirom；Alismone；1- (4- aminomethyl phenyls) ethyl ketone； (3E)-4- (2,6,6- trimethyl-1- cyclohexene-1- bases)-3- butene-2 -one；(3E) -4- (2,6,6- trimethyl -2- hexamethylenes Alkene-1- bases)-3- butene-2 -one；Benzyl carbinol；2H-1- chromen-2-ones；4-methoxybenzaldehyde；10- undecylene aldehydes；Third Acid, phenyl methyl ester；Beta-methyl fenipentol；1,1- diethoxy -3,7- dimethyl -2,6- octadienes；Alpha, alpha-dimethyl benzene second Alcohol；(2E)-1- (2,6,6- trimethyl-1- cyclohexene-1- bases)-2- butene-1 -one；Acetic acid, phenyl methyl ester；Hexamethylene third Acid, 2- propylene base esters；Caproic acid, 2- propylene base esters；1,2- dimethoxy-4 's-(2- acrylic) benzene；The ring of 1,5- dimethyl-two [3.2.1] octyl- 8- ketoximes；4- (4- hydroxy-4-methyls pentyl) -3- cyclohexene-1-formaldehydes；3- butene-2 -ol；2-[[[2,4 (or 3,5)-dimethyl -3- cyclohexenyl group -1- bases] methylene] amino] benzoic acid, methyl ester；- 1- the ketone of 8- rings 16；Crystal violet Sieve ketone；2,6- dimethyl -7- octen-2-ols；2- methoxyl groups -4- (2- acrylic) phenol；- 2,6- is pungent for (2E) -3,7- dimethyl Diene -1- alcohol；2- hydroxy-benzoic acids, (3Z) -3- hexene base esters；2- tridecylene nitriles；4- (2,2- dimethyl -6- methylenecyclohex Base)-3- methyl-3- butene-2 -one；Tetrahydrochysene -4- methyl -2- (2- methyl-1-propylenes base) -2H- pyrans；Acetic acid, (2- methyl fourths Epoxide)-, 2- propylene base esters；Benzoic acid, 2- hydroxyls-, 3- methyl butyl esters；2- butene-1 -one, 1- (2,6,6- trimethyl-1- Cyclohexene -1- bases) -, (Z) -；Cyclopentane-carboxylic acid, 2- hexyl -3- oxos -, methyl ester；Benzenpropanal, 4- ethyl-α, alpha, alpha-dimethyl Base-；3- cyclohexene-1-formaldehydes, 3- (4- hydroxy-4-methyls amyl group)-；Ethyl ketone, 1- (2,3,4,7,8,8a- hexahydro -3,6,8,8- Tetramethyl -1H-3a, 7- methyl alcohol azulene -5- bases) -, [3R- (3. α, 3a. β, 7. β, 8a. α)] -；The hendecanal, 2- methyl- 2H- pyran-2-ones, 6- butyl tetrahydrochysene-；Benzenpropanal, 4- (1,1- dimethyl ethyls)-α .- methyl-；2 (3H)-furanones, 5- Heptyl dihydro-；Benzoic acid, 2- [(7- hydroxyl -3,7- dimethyl octamethylene) amino] -, methyl；Benzoic acid, 2- hydroxyls-, phenyl Methyl ester；Naphthalene, 2- methoxyl groups-；2- cyclopentene-1-ones, 2- hexyls-；2 (3H)-furanones, 5- hexyls dihydro-；Oxirane Carboxylic acid, 3- methyl -3- phenyl -, ethyl ester；2- oxabicyclos [2.2.2] octane, 1,3,3- trimethyl-；Fenipentol, γ .- first Base-；3- octanols, 3,7- dimethyl-；3,7- dimethyl -2,6- octadiene nitriles；3,7- dimethyl -6- octen-1-ols；Terpineol Acetic acid esters；2- methyl -6- methylene -7- octen-2-ols, dihydro derivative；3a, 4,5,6,7,7a- hexahydro -4,7- methyl alcohol -1H- Indenes -6- phenol propionic esters；3-M2BOL acetic acid esters；(Z)-blatter alcohol acetic acid esters；2- ethyl -4- (2,2,3- Trimethyl -3- cyclopentene -1- bases) -2- butene-1-ols；4- (octahydro -4,7- methyl alcohol -5H- Asias indenes -5- bases)-butyraldehyde；3-2,4- Dimethyl-cyclohex alkene -1- formaldehyde；1- (1,2,3,4,5,6,7,8- octahydro -2,3,8,8- tetramethyl -2- naphthyls)-ethyl ketone；2- hydroxyls Base-benzoic acid, methyl ester；2- hydroxy-benzoic acids, hexyl ester；2- phenoxy-ethanols；2- hydroxy-benzoic acids, amyl group ester；2,3- Heptane diketone；2- hexen-1-ols；6- octen-2-ols, 2,6- dimethyl-；Damascone (α, beta, gamma or δ or its mixture), 4,7- Methyl alcohol -1H- indenes -6- phenol, 3a, 4,5,6,7,7a- hexahydros -, acetic acid esters；9- undecylene aldehydes；8- undecylene aldehydes；Isocyclocitral； Ethyl ketone, 1- (1,2,3,5,6,7,8,8a- octahydro -2,3,8,8- tetramethyl -2- naphthyls) -；3- cyclohexene-1-formaldehydes, 3,5- bis- Methyl-；3- cyclohexene-1-formaldehydes, 2,4- dimethyl-；1,6- octadiene -3- alcohol, 3,7- dimethyl -；1,6- octadienes -3- Alcohol, 3,7- dimethyl-, acetic acid esters；Lilial (p-t-Bucinal), and cyclopentanone, 2- [2- (4- methyl -3- cyclohexene -1- Base) propyl group]-and 1- methyl -4- (1- methyl ethylenes) cyclohexene and its mixture.
On the one hand, said composition can include encapsulated perfume particle, and the particle includes water soluble hydroxy compound Or carbamide or modified polyvinyl alcohol.On the one hand, capsule compound includes (a) at least partly water-soluble solid-based Matter, comprising one or more water soluble hydroxy compound, preferred starch；And the perfumery oil that (b) is encapsulated by the solid matrix.
On the other hand, the spices can be pre-composite with polyamines (preferably polyethyleneimine), to form schiff bases (Schiff base)。
Polymer- said composition can include one or more polymer.Example be carboxymethylcellulose calcium, it is poly- (vinyl- Pyrrolidones), PEG, poly- (vinyl alcohol), poly- (vinylpyridine-N-oxide), poly- (vinyl imidazole), polycarboxylic acids Ester (such as polyacrylate), maleic acid/acrylic copolymer and lauryl methacrylate/acrylic copolymer.
Said composition can include one or more amphiphilic cleaning polymer, such as with the chemical combination of following universal architecture Thing：Double ((C2H5O)(C2H4O)n)(CH3)-N+-CxH2x-N+-(CH3)-bis- ((C2H5O)(C2H4O) n), wherein n=is from 20 to 30, And x=is from 3 to 8, or its sulphation or sulfonation variant.
Said composition can include amphiphilic alkoxylate grease cleaning polymer, these polymer have balance it is hydrophilic and Hydrophobic property so that they remove fat particles from fabric and surface.Amphiphilic alkoxylate grease cleaning polymer of the invention Specific embodiment include a core texture and multiple Alkoxylated groups for being connected with that core texture.These can be wrapped The poly- olefin(e) imines (polyalkylenimine) of alkoxylate is included, preferably with interior poly-ethylene oxide block and outer polycyclic oxygen Propane block.
Here, alkoxylate polycarboxylate (for example from polyacrylate prepare those) can be used to provide other oil Fat removal capacity.Such material is described in WO 91/08281 and PCT 90/01815.Chemical Shangdi, these materials include poly- Acrylate, has an ethoxy side chain per 7-8 acrylate unit.Side chain has chemical formula-(CH2CH2O)m(CH2)nCH3, wherein m is 2-3 and n is 6-12.Side chain is that ester is connected to polyacrylate " main chain ", to provide " comb shape " polymer Type structure.Molecular weight with difference, but can be typically in 2000 to 50, in the range of 000.The polycarboxylic acids of such alkoxylate Ester can include composition in this from 0.05wt% to 10wt%.
Surfactant derived from isoprenoid of the invention, and they with other cosurfactants and other The mixture that adjuvant component is formed together is particularly suitable for being used together with amphipathic graft copolymer, preferably the amphipathic stem-grafting copolymerization Thing includes (i) polyethylene glycol backbone；And (ii) and at least one pendant part, the pendant part is selected from polyvinyl acetate Ester, polyvinyl alcohol and its mixture.Preferred amphipathic graft copolymer is the Sokalan HP22 supplied by BASF.Suitable Polymer includes random graft copolymer, the polyethylene oxide copolymer that preferably polyvinyl acetate is grafted, with polycyclic oxygen second Alkane main chain and multiple polyvinyl acetate ester side chain.The molecular weight of PEO main chain is preferably 6000, and PEO Weight ratio with polyvinyl acetate is 40 to 60, and every 50 ethylene oxide unit(s)s have not more than 1 grafting site.
Carboxylate polymer-Composition of the invention also include one or more carboxylate polymer, for example maleate/ Acrylate randomcopolymer or polyacrylate homopolymers.On the one hand, the carboxylate polymer is that have from 4,000Da extremely The polyacrylate homopolymers of 9,000Da or the molecular weight from 6,000Da to 9,000Da.
Dirt release polymer-Composition of the invention can also include one or more dirt release polymer, these Polymer has such as by one of following structure (I), (II) or (III) defined structure：
(I)-[(OCHR1-CHR2)a-O-OC-Ar-CO-]d
(II)-[(OCHR3-CHR4)b-O-OC-sAr-CO-]e
(III)-[(OCHR5-CHR6)c-OR7]f
Wherein：
A, b and c are from 1 to 200；
D, e and f are from 1 to 50；
Ar is the phenylene of 1,4- substitutions；
SAr is the phenylene of 1,3- substitutions, and the phenylene is on 5 by SO3Me replaces；
Me is Li, K, Mg/2, Ca/2, Al/3, ammonium, single-, two-, three-or tetra-allkylammonium, and wherein alkyl is C1-C18Alkyl Or C2-C10Hydroxy alkyl, or its mixture；
R1、R2、R3、R4、R5And R6Independently selected from H or C1-C18N- or iso- alkyl；And
R7It is the C of straight or branched1-C18Alkyl, or straight or branched C2-C30Alkenyl, or with 5 to 9 carbon atoms Cycloalkyl, or C8-C30Aryl, or C6-C30Aryl alkyl.
Suitable soil release polymer is polyester soil release polymers, such as Repel-o-tex polymer, including Repel-o- Tex, SF-2 and SRP6, are supplied by Luo Diya (Rhodia).Other suitable soil release polymers include Texcare polymer, bag Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325 are included, by Clariant (Clariant) supply.Other suitable soil release polymers are Marloquest polymer, for example Marloquest SL, by Sa Sol (Sasol) is supplied.
Cellulosic polymer- composition of the invention also includes one or more cellulosic polymer, including selected from alkyl Those of cellulose, alkyl alkoxy alkylcellulose, carboxyalkyl cellulose, alkylcarboxyalkyl cellulose.On the one hand, Cellulosic polymer is selected from the group, and the group includes carboxymethylcellulose calcium, methylcellulose, methyl hydroxyl ethyl cellulose, methyl Carboxymethylcellulose calcium and its mixture.On the one hand, carboxymethylcellulose calcium have from 0.5 to 0.9 degree of substitution by carboxymethyl and Molecular weight from 100,000Da to 300,000Da.
Enzyme-said composition can include providing one or more enzyme of clean-up performance and/or fabric care benefits.Suitable The example of enzyme includes but is not limited to hemicellulase, peroxidase, protease, cellulase, zytase, lipase, phosphorus Lipase, esterase, cutinase, pectase, mannonase pectin lyase, keratinase, reductase, oxidizing ferment, phenol oxidation Enzyme, lipoxygenase, lignoenzyme, amylopectase, tannase, poly-pentose enzyme, horse traction receive enzyme (malanase), beta glucan Enzyme, arabinosidase, hyaluronidase, chondroitinase, laccase, chlorophyllase and amylase or its mixture.Typical group Conjunction is enzymatic mixture, can be comprising such as protease and lipase together with amylase.When present in the composition, it is foregoing in addition Enzyme can based on the weight of composition with from 0.00001wt% to 2wt%, from 0.0001wt% to 1wt% or from The level of 0.001wt% to 0.5wt% zymoproteins is present.
In general, enzyme viability selected by one or more should (that is, optimal pH, with other compatible with selected detergent Compatibility of enzyme and non-enzyme component, etc.), and one or more enzyme should exist with effective dose.
Cellulase：Suitable cellulase includes those of bacterium or originated from fungus.Including chemical modification or albumen The variant of matter engineering.Suitable cellulase is included from the cellulase of subordinate：Bacillus, pseudomonas, Humicola, Fusarium, Thielavia, acremonium, such as US 4435307, US 5648263, US 5691178, US The fungi produced by Humicola insolens, thermophilic fungus destroyed wire and Fusarium oxysporum disclosed in 5776757 and WO 89/09259 Cellulase.
Particularly suitable cellulase is alkalescence or neutral cellulase with Color care benefit.Such cellulose The example of enzyme is EP 0495257, EP 0531372, WO 96/11262, WO 96/29397, the fibre described in WO 98/08940 The plain enzyme of dimension.Other examples are cellulase variants, such as WO 94/07998, EP 0531315, US 5457046, US 5686593, Those described in US 5763254, WO 95/24471, WO 98/12307 and PCT/DK 98/00299.
Commercially available cellulase includes CelluzymeTMAnd CarezymeTM(Novozymes Company (Novozymes A/ S))、ClazinaseTMAnd Puradax HATM(international corporation of Jie Neng sections (Genencor International )) and KAC-500 (B) Inc.TM(Kao Corp (Kao Corporation)).
On the one hand, preferred enzyme will be including protease.Suitable protease includes bacterium, fungi, plant, viral or dynamic Those of thing origin, such as plant or microbial origin.Preferred microorganism is originated.Including chemical modification or protein engineered Variant.It can be alkali protease, such as serine protease or metalloproteinases.Serine protease may, for example, be S1 families (such as trypsase) or S8 families (such as subtilopeptidase A).Metalloproteinases may, for example, be from such as family The thermolysin of M4 or other metalloproteinases, such as those from M5, M7 or M8 family.
Term " novel subtilases " refers to according to Si Aisen (Siezen) et al., protein engineering (Protein Engng.) 4 (1991) 719-737 and Si Aisen et al., the 501-523's of protein science (Protein Science) 6 (1997) Serine protease subgroup.Serine protease is to be characterized as thering is the silk ammonia with substrate formation covalent adduct in avtive spot One subgroup of the protease of acid.Novel subtilases can be divided into 6 sub-portions, i.e. subtilopeptidase A family, thermophilic egg White enzyme family, Proteinase K family, Lantibiotic peptidase family, Kexin families and Pyrolysin families.
The example of novel subtilases is derived from those of bacillus, for example, be described in US 7262042 and WO Bacillus lentus, Alkaliphilic bacillus, bacillus subtilis in 2009/021867, bacillus amyloliquefaciens, short and small gemma Bacillus and bacillus gibsonii；With subtilopeptidase A slow (lentus), the hay bacillus being described in WO 89/06279 Protease promise and (Novo), subtilopeptidase A Carlsberg (Carlsberg), bacillus licheniformis, subtilopeptidase A BPN ', subtilopeptidase A 309, subtilopeptidase A 147 and subtilopeptidase A 168 and it is described in (WO 93/ 18140) the protease P D138 in.Other useful protease can be described in WO 92/175177, WO 01/016285, Those in WO 02/026024 and WO 02/016547.The example of trypsin like proteases is trypsase (such as pig Or Niu Laiyuan) and Fusarium protease (being described in WO 89/06270, WO 94/25583 and WO 05/040372), and Chymotrypsin derived from cellulomonas cartae (Cellumonas) (is described in WO 05/052161 and WO 05/052146 In).
Further preferred protease is the alkali protease from bacillus lentus DSM 5483 (such as in such as WO Described in 95/23221) and its variant (in WO 92/21760, WO 95/23221, EP 1921147 and EP 1921148 Description).
The example of metalloproteinases is as being described in WO 07/044993 (international corporation of Jie Neng sections (Genencor Int.)) In metalloprotease, for example from bacillus amyloliquefaciens those.
The example of useful protease is the variant in the following：WO 92/19729、WO 96/034946、WO 98/20115、WO 98/20116、WO 99/011768、WO 01/44452、WO 03/006602、WO 04/03186、WO 04/ 041979th, WO 07/006305, WO 11/036263, WO 11/036264, especially in one or more of following position Variant with substitution：3、4、9、15、27、36、57、68、76、87、95、96、97、98、99、100、101、102、103、104、 106、118、120、123、128、129、130、160、167、170、194、195、199、205、206、217、218、222、224、 232nd, 235,236,245,248,252 and 274, use BPN ' to number.It is highly preferred that these Subtilase variants can be wrapped Containing following mutation：S3T、V4I、S9R、A15T、K27R、*36D、V68A、N76D、N87S,R、*97E、A98S、S99G,D,A、 S99AD、S101G,M,R S103A、V104I,Y,N、S106A、G118V,R、H120D,N、N123S、S128L、P129Q、 S130A、G160D、Y167A、R170S、A194P、G195E、V199M、V205I、L217D、N218D、M222S、A232V、 K235L, Q236H, Q245R, N252K, T274A (use BPN ' to number).
Suitable commercially available protease includes those sold with following trade name：DuralaseTm、 DurazymTm、Ultra、Ultra、Ultra、With(Novi believes Company), those sold with following trade name： Purafect PreferenzTm、PurafectPurafectPurafectEffectenzTm、 And(Danisco/E.I.Du Pont Company (Danisco/DuPont)), AxapemTm(Ji Site Buro Cadizs are public Department (Gist-Brocases N.V.)), BLAP (sequence is shown in Figure 29 of US 5352604) and its variant (Henkel share (Henkel AG)) and KAP (Alkaliphilic bacillus subtilopeptidase A) from Kao Corp (Kao).
Suitable lipase and cutinase include those of bacterium or originated from fungus.Including chemical modification or protein engineering The mutant enzyme of change.Example includes the lipase from thermophilic fungal category, for example, be such as described in EP 258068 and EP 305216 In from Thermomyces lanuginosus (being named as thin cotton like humicola lanuginosa previously)；Cutinase from Humicola, such as it is special Different humicola lanuginosa (WO 96/13580)；(some in these are renamed as Bai Ke to the lipase of the bacterial strain from pseudomonas now Hall Bordetella), such as Pseudomonas alcaligenes or pseudomonas pseudoalcaligenes (EP 218272), Pseudomonas cepacia (EP 331376), pseudomonas strain SD705 (WO 95/06720＆WO 96/27002), Wisconsin pseudomonad (P.wisconsinensis)(WO 96/12012)；GDSL- type streptomyces lipase (WO 10/065455)；From rice blast The cutinase (WO 10/107560) of germ；Cutinase (US 5,389,536) from pseudomonas mendocina；From brown Thermophilic lipase (WO 11/084412, WO 13/033318, the WO 2013/ for splitting spore bacterium (Thermobifida fusca) 096653)；Geobacillus stearothermophilus lipase (WO 11/084417)；Lipase (WO from bacillus subtilis 11/084599)；And from streptomyces griseus (WO 11/150157) and rotation streptomycete (S.pristinaespiralis) The lipase of (WO 12/137147).
Other examples are lipase Variants, for example, be described in EP 407225, WO 92/05249, WO 94/01541, WO 94/25578、WO 95/14783、WO 95/30744、WO 95/35381、WO 95/22615、WO 96/00292、WO 97/ 04079th, WO 97/07202, WO 00/34450, WO 00/60063, WO 01/92502, WO 07/87508 and WO 09/ Those in 109500.
Preferred commercialization lipase product includes LipolaseTM、LipexTM；LipolexTMAnd LipocleanTM(Novi Letter company), Lumafast (initially coming from Genencor Company (Genencor)) and Lipomax (initially come from Ji Site-Bock De Si companies (Gist-Brocades)).
Other examples are the lipase of sometimes referred to as acyltransferase or Perhydrolase again, such as with antarctic candida (Candida antarctica) lipase A have homology acyltransferase (WO 10/111143), from shame dirt branch Acyltransferase (WO 05/56782), the Perhydrolase from the families of CE 7 of bacillus (Mycobacterium smegmatis) The variant of (WO 09/67279) and M. smegmatis perhydrolase (steps the textile limited public affairs of dyeization especially from Hensel Take charge of S54V used in the commercial product Gentle Power Bleach of (Huntsman Textile Effects Pte Ltd) Variant) (WO 10/100028).
On the one hand, other preferred enzymes include showing inscribe-β-Isosorbide-5-Nitrae-dextranase activity derived from microorganism Endoglucanase (EC3.2.1.4), including endogenous bacterial peptide for the member of bacillus, with US Amino acid sequence SEQ ID NO in 7141403:2 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%th, the sequence of 99% or even 100% uniformity, and its mixture.Suitable endoglucanase is in trade (brand) nameWithSold under (Novozymes Company).
Other preferred enzymes are included in trade (brand) nameUnder the fruit sold Glue lyases, and in trade (brand) nameUnder the mannase (Novozymes Company) sold, and(Danisco/E.I.Du Pont Company (Danisco/DuPont)).
One or more detergent enzyme can include one or more single additive of enzyme by addition, or pass through Addition includes the combined additive of all these enzymes and is included in detergent composition.Detergent additives of the invention, That is, independent additive or combined additive, can be configured to, for example particle, liquid, slurry etc..Preferred detergent addition Agent preparation is particle, especially non-dust particles；Liquid, especially stabilized liquid；Or slurry.
Non-dust particles can be manufactured for example as disclosed in US 4106991 and US 4661452, and can be optionally It is coated with by method as known in the art.The example of waxy coating materials be mean molecule quantity be 1000 to 20000 it is poly- (oxirane) product (polyethylene glycol, PEG)；With the 16-50 ethoxylated nonylphenol of ethylene oxide unit(s)；With 15 to 80 ethoxylized fatty alcohols of ethylene oxide unit(s), wherein alcohol contain 12 to 20 carbon atoms；Fatty alcohol；Aliphatic acid；With And the list of aliphatic acid-and double-and glyceryl ester.Example suitable for the film-forming coating materials by fluidization application exists Be given in GB1483591.Liquid enzyme formulation for example can add polyalcohol (such as propane diols), sugar by according to the method established Or sugar alcohol, lactic acid or boric acid and stabilize.Shielded enzyme can be prepared according to the method disclosed in EP238216.
Dye transfer inhibitor-Composition of the invention can also include one or more dye transfer inhibitor.Properly Polymeric dye transfer inhibitor include but is not limited to polyvinyl pyrrolidone polymers, polyamines N- oxide polymers, N- The copolymer of vinylpyrrolidone and N- vinyl imidazoles, Ju Yi Xi oxazolidones and polyvinyl imidazole or its mixture.When When being present in composition, dye transfer inhibitor can by from 0.0001wt% to 10wt%, from 0.01wt% to 5wt% or Level from 0.1wt% to 3wt% is present.
Brightener- composition of the invention can also include other component, and these components to the article of positive cleaning Color, such as brightener.
Said composition can include C.I. brighteners 260, in the alpha-crystal form with following structure：
On the one hand, brightener is cold water solubles brightener, for instance in the C.I. brighteners of alpha-crystal form 260.On the one hand, brightener is mostly in alpha-crystal form, it means that typically at least 50wt%, at least 75wt%, extremely Few 90wt%, at least 99wt% or even substantially all of C.I. brighteners 260 are in alpha-crystal form.
Brightener is typically in micronized particulate form, with from 3 to 30 microns, from 3 microns to 20 microns or from 3 to 10 microns of weighted average primary particle size.
Said composition can include the C.I. brighteners 260 in β-crystal form, and (i) is in alpha-crystal shape The weight ratio of C.I. brighteners 260 that the C.I. brighteners 260 of formula are in β-crystal form with (ii) can be to Few 0.1 or at least 0.6.BE 680847 is related to the method for the C.I. brighteners 260 in alpha-crystal form to be obtained.
The commercial optical brightener that can be used in the present invention can be divided into multiple subgroups, and these subgroups include but are not It is necessarily limited to：Talan, pyrazoline, cumarin, carboxylic acid, methine cyanines, dibenzothiophenes -5,5- dioxide, azoles, 5 With the derivative of 6 membered ring heterocyclics and other mix agent.The example of such brightener be disclosed in " production of brightener and should With ", M. Zuo Helaodenike (Zahradnik), by John Wiley father and son company (John Wiley＆Sons), New York (1982) Publish.The specific non-limiting examples that can be used for the optical brightener of the present composition are in US 4790856 and US Those identified in 3646015.
Suitable brightener has following structure in addition：
Suitable brightener level is included from 0.01wt%, from 0.05wt%, from 0.1wt% or from 0.2wt%'s Reduced levels to 0.5wt% or 0.75wt% higher level.
On the one hand, brightener can be loaded on clay to form particle.Silicate-composition of the invention can be with Comprising silicate, such as sodium metasilicate or potassium.Said composition can be included from 0wt% to less than 10wt% silicate, to 9wt%, Or to 8wt% or to 7wt% or to 6wt% or to 5wt% or to 4wt% or to 3wt% or even to 2wt%, and And from higher than 0wt% or the silicate from 0.5wt% or from 1wt%.Suitable silicate is sodium metasilicate.
Dispersant- composition of the invention can also include dispersant.Suitable water-soluble organic materials include homopolymerization Combined polymerization acid or its salt, wherein polycarboxylic acids include at least two carboxyls, the two carboxyls by no more than two carbon atoms that This is separated.
Enzyme stabilizers- enzyme for using in the composition can be by various technologies come stabilization.Enzyme as used herein can With the presence by calcium and/or the water-soluble source of magnesium ion come stabilization.The example of conventional stabilizer is, for example, propane diols Or glycerine, sugar or sugar alcohol, lactic acid, boric acid or boronic acid derivatives, such as aromatic boric acid ester, or phenyl boronic acid derivative, for example 4- formylphenyl boronic acids, and can be as prepared said composition described in such as WO 92/19709 and WO 92/19708. In the case of waterborne compositions comprising protease, reversible protease inhibitors can be added, such as including borate, 4- formyls The boron compound of base phenylboric acid, phenylboric acid and its derivative, or such as chemical combination of calcium formate, sodium formate and 1,2-PD Thing, further to improve stability.
Solvent- suitable solvent includes water and other solvents, such as lipophilic fluid.The example of suitable lipophilic fluid Including siloxanes, other silicone, hydrocarbon, glycol ether, glycerol derivatives (such as glycerin ether), fluoridized amine, it is fluoridized and Hydrofluoroether solvent, the organic solvent of low volatility nonfluorinated, diol solvent, other environmentally friendly solvents and its mixture.
Structural agent/thickener- structuring liquid can from internal structured, thus structure by primary sector (for example, table Face surfactant material) formed, and/or provided by using secondary component (for example, polymer, clay and/or silicate material) Three dimensional matrix structure and from external structurant.Said composition can include from 0.01wt% to 5wt% or from 0.1wt% to The structural agent of 2.0wt%.The structural agent is typically chosen from the following group, and the group is made up of the following：Diglyceride and glycerine Three esters, stearic acid diethylene glycol dilaurate, microcrystalline cellulose, the material based on cellulose, microfibrous cellulose, the alkalescence of hydrophobically modified Expandable emulsion (such as Polygel W30 (3VSigma)), biopolymer, xanthans, gellan gum and its mixture. Suitable structural agent includes the castor oil and its unethoxylated derivative of hydrogenation.Suitable structural agent is disclosed in US In 6855680.Such structural agent has screw thread spline structure system, and the system has a series of aspect ratios.Other are suitable Structural agent and it is described in WO 10/034736 for their method to be obtained.
Conditioning agent- composition of the invention can include hard fat compound.Useful hard fat herein Compound has 25 DEG C or fusing point higher, and is selected from the group, and the group is made up of the following：Fatty alcohol, aliphatic acid, fatty alcohol Derivative, derivative of fatty acid and its mixture.Such compound with low melting point is not intended to be incorporated herein part In.The non-limiting examples of high melting compound are found in international cosmetic ingredient dictionary, the 5th edition, and 1993, and CTFAization Cosmetic Ingredient Dictionary, the second edition, in 1992.
In view of it is (wet and slippery sense such as during wet hair is applied to, soft and send out dry to provide improved regulation benefit Moisturizing sense), hard fat compound by with from 0.1wt% to 40wt%, from 1wt% to 30wt%, from 1.5wt% to 16wt%, the level from 1.5wt% to 8wt% are included in the composition.
Composition of the invention can include cationic polymer.The concentration of cationic polymer is typically in the composition Scope is from 0.05wt% to 3wt%, from 0.075wt% to 2.0wt% or from 0.1wt% to 1.0wt%.Used in expection Under the pH of said composition, suitable cationic polymer will with least 0.5meq/gm, at least 0.9meq/gm, at least 1.2meq/gm, at least 1.5meq/gm or the cationic charge density less than 7meq/gm and less than 5meq/gm, the pH's Scope will be substantially from pH 3 to pH 9 or between pH 4 and pH 8.Here, " cationic charge density " of polymer is The ratio between molecular weight of positive charge number and polymer on finger polymer.The mean molecule quantity of such suitable cationic polymer Will be substantially between 10,000 and 10,000,000, between 50,000 and 5,000,000 or between 100,000 and 3,000,000.
Suitable cationic polymer for using in the present compositions includes cation nitrogen moiety, for example Quaternary ammonium or cation protonated amino part.Any anionic counter ion can be associated with cationic polymer and used, only Want polymer to be held in solution in, in composition or in the condensed phase of composition, as long as and counter ion in physics and chemistry The upper main component with composition mutually perhaps will not inadequately damage composition properties, stability or U.S. otherwise Sense.The non-limiting examples of such counter ion include halide (for example, chloride, fluoride, bromide, iodide), sulfuric acid Salt and Methylsulfate.
The non-limiting examples of such polymer are described in CTFA cosmetic ingredient dictionaries, the third edition, Estlin (Estrin), Crosley (Crosley) and Haynes (Haynes) write (cosmetics, toiletry and perfume joint public affairs Department (The Cosmetic, Toiletry, and Fragrance Association, Inc.), Washington (1982)).
Other suitable cationic polymers for using in the composition include polysaccharide polymer, cation Guar Bean gum derivative, the cellulose ether of quaternary nitrogen containing, synthetic polymer, the copolymer of etherified cellulose, guar gum and starch.When When use, cationic polymer in this is dissolvable in water in composition or is dissolvable in water the complex coacervation phase in composition In, the condensed phase is cationic polymer and anion, both sexes and/or zwitterionic surface-active agent component by mentioned earlier Formed.The complex coacervation thing of cationic polymer can also be formed with other electrically charged materials in composition.Suitable sun from Sub- polymer is described in US 3962418；US 3958581；In US 2007/0207109.
Composition of the invention can be included as the non-ionic polymers of conditioning agent.There is the molecule more than 1000 herein The polyglycols (polyalkylene glycol) of amount is useful.Those with below general formula are useful：
Wherein R95It is selected from the group, the group is made up of the following：H, methyl and its mixture.Conditioning agent, and particularly Silicone, can be included in the composition.Typically comprise to form emulsifying liquid for the conditioning agent in composition of the invention The water-insoluble of particle, water dispersible, non-volatile liquid.Suitable conditioning agent for using in the composition is logical Often it is characterized as those conditioning agents of following item：Silicone (for example, silicone oil, cation silicone, silicone adhesive, refractiveness silicone high, with And silicone resin), organic regulation oil (for example, hydrocarbon ils, polyolefin and fatty ester) or its combination, or exist in another manner Those conditioning agents of liquid dispersion particle are formed in this aqueous tenside matrix.Such conditioning agent should in physics and It is chemically compatible with the key component of composition, and should not inadequately damages composition stabilization otherwise Property, aesthetic feeling or performance.
The concentration of conditioning agent should be enough to provide desired regulation benefit in the composition.This concentration can be with tune Section agent, desired regulation performance, the mean size of conditioning agent particle, the type of other components and concentration and other it is similar because Element and change.
The concentration range of silicone conditioning agent is typically from 0.01wt% to 10wt%.Suitable silicone conditioning agent and right The U.S. Reissue patent No. 34,584 is described in the non-limiting examples of the optional suspending agent of silicone；US 5104646；US 5106609；US 4152416；US 2826551；US 3964500；US 4364837；US 6607717；US 6482969；US 5807956；US 5981681；US 6207782；US 7465439；US 7041767；US 7217777；US 2007/ 0286837 A1；US 2005/0048549 A1；US 2007/0041929 A1；GB 849433；In DE 10036533, by institute There is document incorporated herein by reference；The chemistry and technology of silicone, New York：Academic press (1968)；General Electric silicone rubber Glue product data list SE 30, SE 33, SE 54 and SE 76；Silicone compounds, Petrarch system house (Petrarch Systems, Inc.) (1984)；And polymer science and engineering encyclopedia, volume 15, second edition, 204-308 pages, John In Willie father and son company (John Wiley＆Sons, Inc.) (1989).
Composition of the invention can also include at least one organic regulation oil conduct regulation from 0.05wt% to 3wt% Agent, combines individually or with other conditioning agents such as silicone (described herein).Suitable regulation oil include hydrocarbon ils, polyolefin, with And fatty ester.It is in US 5674478 and US 5750122 or in US 4529586 to be suitable also in composition in this； US 4507280；US 4663158；US 4197865；US 4217914；US 4381919；And described in US 4422853 Conditioning agent.
Hygiene and cacogeusia- composition of the invention can also include ricinoleic acid zinc, thymol, quaternary ammonium salt (for example), polyethyleneimine is (such as from BASF) and its zinc complexes, silver and silver compound are (especially It is designed to slowly discharge Ag+Or those of nanometer silver dispersions) in one or more.
Probiotic- these compositions can include probiotic, in be described in WO 09/043709.
Foam improverIf-foaming high is desirable to, foam improver (such as C10-C16Alkanolamide or C10-C14Alkylsurfuric acid Ester) can typically with the level incorporation composition of 1wt% to 10wt%.C10-C14Monoethanol and diglycollic amide are elaborated The typical classification of such foam improver.Such foam improver is with foaming adjuvant surfactant high (for example, above-mentioned oxidation Amine, glycine betaine and sulfobetaines (sultaine)) it is used together and is also advantageous.If desired, water soluble magnesium And/or calcium salt (such as MgCl2、MgSO4、CaCl2、CaSO4Deng) typically can be added with the level of 0.1wt% to 2wt%, To provide other foam and to strengthen grease removal capacity.
Foam in hibitors- the compound for reducing or suppressing formation of foam can be mixed in composition of the invention.Bubble Foam suppresses in such as so-called " the high concentration cleaning procedure " described in US 4489455 and US 4489574 and in front bearing formula (front-loading-style) it is probably especially important in rinsing maching.Diversified material can serve as foam suppression Agent, and foam in hibitors is for a person skilled in the art well known.See, e.g. Ke Keaosimo chemical industry hundred Section's pandect (Kirk Othmer Encyclopedia of Chemical Technology), the third edition, volume 7,430- Page 447 (John Wiley father and son company, 1979).The example of foam in hibitors includes mono carboxylic aliphatic acid and therein solvable Salt, high-molecular-weight hydrocarbons such as paraffin, fatty acid ester (for example, fatty acid triglycercide), the fatty acid ester of monovalent alcohol, aliphatic C18-C40Ketone (such as stearone), the amino triazine of N- alkylations, preferably with the wax hydrocarbon of the fusing point under about 100 DEG C, silicone Foam in hibitors, and secondary alcohol.Foam in hibitors is described in US 2954347；US 4265779；US 4265779；US 3455839；US 3933672；US 4652392；US 4978471；US 4983316；US 5288431；US 4639489；US 4749740；US 4798679；US 4075118；EP 89307851.9；EP 150872；And in DOS 2,124,526.
For there is any detergent composition being ready to use in automatic washing machine, foam should not form their spillings and wash Wash the degree of machine.When deployed, foam in hibitors is preferably what is existed with " foam amount of suppression "." foam amount of suppression " means combination The makers-up of thing can select the amount of this foam controller, and this amount will fully control foam to cause for automatic laundry Low foaming laundry detergent compositions in machine.
Composition in this will generally comprise the foam in hibitors from 0wt% to 10wt%.When as foam in hibitors, Mono carboxylic aliphatic acid and salt therein typically will exist with the amount of up to 5wt%.Preferably, using from 0.5wt% to Aliphatic mono-carboxylic acids' ester foam in hibitors of 3wt%.Silicone foam inhibitor is typically used with the up to amount of 2.0wt%, although Amount higher can be used.Generally suppressed using single stearoyl phosphate foam with the amount of the scope from 0.1wt% to 2wt% Agent.Hydrocarbon foam in hibitors is typically used with the amount of the scope from 0.01wt% to 5.0wt%, although water higher can be used It is flat.Alcohol foam in hibitors is typically used with 0.2wt% to 3wt%.
Composition in this can have cleaning action in the range of pH wide.In certain embodiments, these compositions With from pH 4 to pH 11.5 cleaning action.In other embodiments, these compositions 11 from pH 6 to pH, from pH 7 to PH 11,11 or 11,5 active from pH 10 to pH 11 from pH 8 to pH, from pH 9 to pH.
Composition in this can have cleaning action in the temperature (such as 90 DEG C from 10 DEG C or more as little as) of wide scope. Preferably, the temperature will be less than 50 DEG C or 40 DEG C or even 30 DEG C.In certain embodiments, for these compositions most Suitable temperature range be from 10 DEG C to 20 DEG C, from 15 DEG C to 25 DEG C, from 15 DEG C to 30 DEG C, from 20 DEG C to 30 DEG C, from 25 DEG C to 35 DEG C, From 30 DEG C to 40 DEG C, from 35 DEG C to 45 DEG C or from 40 DEG C to 50 DEG C.
The form of composition
Composition described here is advantageously utilised in such as laundry applications, hard-surface cleaning, dishwashing detergent application, together with In cosmetic applications (such as artificial tooth, tooth, hair and skin).Composition of the invention be specifically solid or cleaning liquid and/or Treatment compositions.On the one hand, the present invention relates to a kind of composition, the form of wherein said composition is selected from the group, the group by with Lower every composition：It is a kind of conventional, compression or concentration liquid；A kind of gel；A kind of cream；A kind of soap bar；It is a kind of conventional Or the powder of compression；A kind of granular solids；Uniform or multilayer tablet of the one kind with two or more layers (identical or different phase) Agent；A kind of bag with one or more rooms；A kind of single or multiple room unit dosage forms；Or its any combinations.
The form of said composition can be by the component in multiple rooms (such as water soluble bag) or in the different layers of tablet It is physically separated from.It is possible thereby to the negative storage between avoiding component interacts.In wash solution, each room Different solubility curves can also cause the delayed dissolved of the component of selection.
Bag can be configured as single or multiple rooms.It can have suitable appearance to hold any form, the shape of said composition Shape and material, such as before being contacted with water, do not allow said composition to be discharged from bag.Bag is by the water-soluble of encapsulation inner volume Property film is made.The inner volume can be divided into the room with bag.Preferred film is the polymeric material to form film or piece, preferably Polymer.Preferred polymer, copolymer or derivatives thereof are selected from polyacrylate and water-soluble acrylic ester copolymer, first Base cellulose, carboxymethylcellulose calcium, dextrin sodium, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malt paste Essence, most preferably polymethacrylates, polyvinyl alcohol copolymer and hydroxypropyl methyl cellulose (HPMC).Preferably, The level of the polymer (such as PVA) in film is at least about 60%.Preferred mean molecule quantity will be typically about 20,000 To about 150,000.Film can also be blend composition, and the blend composition includes degradable and water soluble and water soluble polymer Blend, such as PLA and polyvinyl alcohol (it is known under trade reference M8630, such as by the MonoSol of Indiana, USA LLC Companies) plus plasticizer, as glycerine, ethylene glycol, propane diols, sorbierite and its mixture.These bags can include solid Laundry cleaning composition or constituent part and/or liquid cleansing composition or the constituent part separated by water-solubility membrane.For liquid The room of body component in composition can be different from the room including solid (A1 of US 2009/0011970).
Water-solubility membrane- composition of the invention can also be encapsulated within water-solubility membrane.Preferably, preferred membrane material It is polymeric material.Membrane material can for example be obtained by the casting of polymeric material, blowing, extrusion or blown extrusion, As known in the art.It is to be selected to be suitable for being used as preferred polymer, copolymer of bag material or derivatives thereof：Poly- second Enol, polyvinylpyrrolidone, polyalkylene oxide, acrylamide, acrylic acid, cellulose, cellulose ether, cellulose esters, cellulose Acid amides, polyvinylacetate, polycarboxylic acids and salt, polyaminoacid or peptide, polyamide, polyacrylamide, maleic acid/acrylic acid common Polymers, polysaccharide (including starch and gelatin), natural gum (such as xanthans and carragheen (carragum)).Preferred polymer Selected from polyacrylate and water-soluble acrylic resin copolymer, methylcellulose, sodium carboxymethylcellulose, dextrin, ethyl cellulose Element, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, polymethacrylates, and most preferably it is selected from poly- second Enol, polyvinyl alcohol copolymer and hydroxypropyl methyl cellulose (HPMC), and combinations thereof.Preferably, polymer is in bag material (example Such as, PVA polymer) in level be at least 60wt%.Polymer can have any weight average molecular weight, preferably from about 1.000 to 1.000.000, from about 10.000 to 300.000, from about 20.000 to 150.000.The mixture of polymer can be with As bag material.
Natively, the film of different membrane materials and/or different-thickness can be used for making room of the invention.It is different in selection Film in benefit be gained room can represent different solubilities or release characteristic.
Preferred membrane material is the known PVA film under MonoSol brand names M8630, M8900, H8779, and is retouched It is set forth in those in US 6166117 and US 6787512, and the PVA film with corresponding solubility and deformation behaviour.
Membrane material in this can also include one or more additive component.For example, can be with it is beneficial that addition plasticising Agent, such as glycerine, ethylene glycol, diethylene glycol, propane diols, sorbierite and its mixture.Other additives include needing to be delivered To the functional detergent additive of slurry, such as organic polymer dispersing agents etc..
The method for manufacturing composition
Composition of the invention can be configured to any suitable form, and can be by selected of the person of being formulated Where prepared by method, and the non-limiting examples of these methods are described in the example of applicant and US 4990280；US 20030087791 A1；US 20030087790 A1；US 20050003983 A1；US 20040048764 A1；US 4762636；US 6291412；US 20050227891 A1；EP 1070115 A2；US 5879584；US 5691297；US 5574005；US 5569645；US 5565422；US 5516448；US 5489392；In US 5486303, all documents are led to Cross to apply and be hereby incorporated by.Composition of the invention or composition prepared in accordance with the present invention include cleaning and/or treatment compositions, Including but not limited to it is used to be processed in fabric and household care field the combination on fabric, hard surface and any other surface Thing, including：Air care (including air freshener and smell delivery system), car care, wash the dishes, fabric regulation it is (including soft Change and/or pure and fresh), laundry de-sludging, laundry and rinsing additive and/or nursing, hard surface cleaning and/or treatment (including ground Plate and Closestool cleanser), particle or powder type is universal or " heavy load " detergent, especially cleaning detergent；Liquid, glue Or the universal detergent of cream form, especially so-called heavy load kind of liquid；Liquid is fine-fabric detergent；Wash the dishes by hand Agent or light load dishwasher detergent, especially those of bubbling type high；Automatic dishwasher agent, including for making for family and public organizations Different tablet, particle, liquid and rinse aid type：Automobile or carpet shampoos, bathroom detergent (including detergent of closet Agent)；And cleaning adjuvant, for example bleach additive and " dirt-cling (stain-stick) " or pretreatment type, dress Carry the composition (for example adding the lamella of drier) of matrix.It is preferably used for cleaning and/or processing textile and/or hard table The composition and method in face (most preferably textile).Composition is preferably in the pre-treatment step of washing process or main The composition for (being most preferably for textile washing step) in washing step and using.
As used herein, term " fabric " and/or hard surface cleaning and/or treatment compositions " are cleaning and treatment group The subset of compound, unless otherwise noted, the subset includes particle or powder type is universal or " heavy load " detergent, especially Cleaning detergent；Liquid, glue or the universal detergent of cream form, especially so-called heavy load kind of liquid；Liquid is finely knitted Thing detergent；Manual dishwasher detergent or light load dishwasher detergent, especially those of bubbling type high；Automatic dishwasher agent, including be used for Different tablets, particle, liquid and the rinse aid type used for family and public organizations；Cleaning liquid and disinfectant, vapour Car or carpet shampoos, bathroom detergent (including Closestool cleanser)；Fabric regulating composition (including softening and/or pure and fresh), can With in liquid, solid and/or drier sheet form；Together with cleaning adjuvant, for example bleach additive and " decontamination rod " or Pretreatment type, the composition (for example adding the lamella of drier) for loading matrix.All of applicable such composition can Be in standard, concentration or even in the form of high enrichment, or even can in some aspects in non-to such composition Aqueous degree.
Application method
Present invention resides in fabric and/or household care field be used for clean any surface (including treatment textile or Crust or other surfaces) method.In one aspect of the invention, the method be included in washing process pre-treatment step or Main wash step (is most preferably in textile washing step using or is alternatively used in dishwashing detergent (including hand Both dynamic and automatic/mechanical dishwashing detergents) in use) in contact pending processing surface the step of.At one of the invention In embodiment, lipase Variant and other components are sequentially added in the method for cleaning and/or processing surface.Can replace Dai Di, simultaneously adds lipase Variant and other components.
As used herein, washing includes but is not limited to clean and mechanical agitation.Washing can be carried out with foam compositions (as described in WO 08/101958), and/or by apply alternating pressure (pressure/vacuum) be used as scouring and it is churned mechanically Addition method or alternative are carried out.Such surface or fabric are dried can adopt by family or industrial environment With any one of universal means complete.Cleasing compositions of the invention are preferably suited for should in laundry and dishwashing detergent Used in.Therefore, the present invention includes the side for cleaning objects (including but not limited to fabric, tableware, cutter and kitchen tools) Method.The method includes making the step of having object to be cleaned to be contacted with the Cleasing compositions, and the Cleasing compositions include application At least one of the Cleasing compositions of people, cleaning additive or its mixture embodiment.Fabric can include can be in routine The most of any fabric being washed under consumer or public organizations' use condition.The solution can have the pH from 8 to 10.5. Composition can be used with the concentration from 500ppm to 15.000ppm in the solution.Water temperature range is typically from 5 DEG C to 90 ℃.The ratio between water and fabric are typically from 1:1 to 30:1.
On the one hand, the present invention relates to be used to produce a kind of method of composition, the polypeptide and SEQ ID using polypeptide NO:2 have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%th, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, Or 100% uniformity.On the one hand, the present invention relates to said composition be used for cleaning objects purposes.
On the one hand, the method the present invention relates to produce composition, the method includes addition and SEQ ID NO:2 have extremely Few 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%th, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% 1 The polypeptide of cause property, and surfactant.On the one hand, the method the present invention relates to be used for clean surface, the method includes making to treat Lipid spot is contacted with the Cleasing compositions present on the surface of cleaning.On the one hand, the present invention relates to be used to hydrolyze presence The method of the lipid in the spot and/or spot on surface, the method includes making spot and/or spot be connect with Cleasing compositions Touch.
Plant
The invention further relates to plant, such as genetically modified plants, plant part or plant cell, these plants include the present invention Polynucleotides, with recyclable amount expression and to produce the polypeptide.The polypeptide can be reclaimed from plant or plant part.Can Alternatively, in statu quo the plant comprising the polypeptide or plant part can be used to improve food or quality of the fodder, for example, changing Kind nutritive value, palatability and the rheological equationm of state, or be used to destroy ANFs.
Genetically modified plants can be dicots (dicotyledon) or monocotyledonous (monocotyledon).Monocotyledon Example be careless such as English grass (bluegrass, Poa L .), forage grass such as Festuca (Festuca), Lolium (Lolium), temperate zone grass such as Agrostis (Agrostis) and cereal for example wheat, oat, rye, barley, paddy rice, sorghum, With maize (corn).
The example of dicotyledon is tobacco, beans (such as lupin (lupins), potato, sugar beet (sugar Beet), pea, beans (bean) and soybean (soybean)) and crucifer (Cruciferae (family Brassicaceae)) (such as cauliflower, rapeseed and the model organism arabidopsis being closely related).
The example of plant part is stem, callus, leaf, root, fruit, seed and stem tuber and including these parts Independent body, for example, epidermis, mesophyll, parenchymal tissue (parenchyme), vascular tissue, separate living tissue.Specified plant cell Compartment, such as chloroplaset, apoplast (apoplast), mitochondria, vacuole, peroxisome and cytoplasm are also considered as planting Thing part.Additionally, any plant cell, either which kind of is tissue-derived, is considered as plant part.Similarly, plant portion Point, such as separate to contribute to the particular organization for utilizing of the invention and cell to be also considered as plant part, for example embryo, endosperm, Aleuron and kind skin.
Be also included in the scope of the invention be such plant, plant part and plant cell filial generation.
The genetically modified plants or plant cell for expressing polypeptide can build according to methods known in the art.In brief, The plant or plant cell are built by the following method：One or more expression constructs of coded polypeptide are incorporated into plant place In key-gene group or Chloroplast gene, and the modified plant or plant cell of gained is set to breed as genetically modified plants or plant Cell.
Method the invention further relates to produce polypeptide of the invention, including：A () is under conditions of the polypeptide is helped to create Culture includes genetically modified plants or the plant cell of the polynucleotides for encoding the polypeptide；And (b) reclaims the polypeptide.
The present invention is further described by following instance, but should not be construed as limitation of the scope of the invention.
Example
Culture medium and solution
Except as otherwise noted, the chemicals as buffer solution and substrate is at least the commodity of reagent grade.Commercially available enzyme LipolaseTMAnd LipexTMAvailable from Novozymes Company.
LipolaseTMIncluding wild type triacylglycerol from the thermophilic hyphomycete of thin cotton like, being expressed in aspergillus oryzae Lipase.
LipexTMIncluding derived from wild type dredge cotton like thermophilic hyphomycete triacylglycerol lipase, with mutation T231R and N233R's and in aspergillus oryzae express triacylglycerol lipase.
Bacterial strain
Using purchased from Tiangeng (TIANGEN) (Tiangeng bio tech ltd (TIANGEN Biotech Co.Ltd.), Beijing, China) Escherichia coli Top-10 bacterial strains breed expression vector.
Using aspergillus oryzae MT3568 bacterial strains be used for coded polypeptide gene heterogenous expression, the polypeptide with have lipase activity The polypeptide of property has homology.Aspergillus oryzae MT3568 is the gene derivative of amdS (acetamidase) destructions of aspergillus oryzae JaL355 (WO 02/40694), wherein recovering pyrG auxotrophies by with pyrG gene disruptions Aspergillus oryzae acetamidase (amdS) gene Type.
Culture medium
YPM culture mediums are by the yeast extract of 10g, the bactopeptone of 20g, the maltose of 20g and complement to The deionized water of 1000ml is constituted.
LB plates are by the chlorination of the bacto-tryptone (Bacto-Tryptone), the yeast extract, 10g of 5g of 10g Sodium, the Bacto agar of 15g and complement to 1000ml deionized water constitute.
LB culture mediums and are complemented to by the sodium chloride of the bacto-tryptone of 10g, the yeast extract of 5g and 10g The deionized water of 1000ml is constituted.
COVE sucrose plates by the sucrose of 342g, the agar powder of 20g, the COVE salting liquids of 20ml and complement to 1 liter go from Sub- water is constituted.Culture medium is carried out into sterilizing 15 minutes under 15psi by autoclaving.By the culture medium be cooled to 60 DEG C and Add acetamide, the CsCl of 15mM, the triton x-100 (50 μ l/500ml) of 10mM.
For separate COVE-2 plate tubes：30g/L sucrose, 20ml/L COVE salting liquids, 10mM acetamides, 30g/L promises Boolean (noble) agar (Difco, catalog number (Cat.No.) 214220).
COVE salting liquids by 26g MgSO4·7H2The KH of KCl, 26g of O, 26g2PO4, 50ml COVE trace metals it is molten Liquid and complement to 1000ml deionized water constitute.
COVE trace metal solutions by 0.04g Na2B4O7·10H2The CuSO of O, 0.4g4·5H2The FeSO of O, 1.2g4· 7H2The MnSO of O, 0.7g4·H2The Na of O, 0.8g2MoO4·2H2The ZnSO of O, 10g4·7H2O and complement to going for 1000ml Ionized water is constituted.
Bacterial strain
Using purchased from Tiangeng (TIANGEN) (Tiangeng bio tech ltd (TIANGEN Biotech Co.Ltd.), Beijing, China) Escherichia coli Top-10 bacterial strains breed expression vector.
Aspergillus oryzae (Aspergillus oryzae) MT3568 bacterial strains are used for heterogenous expression.Aspergillus oryzae MT3568 is meter Qu The gene derivative (WO 02/40694) of amdS (acetamidase) destructions of mould JaL355, wherein by using pyrG gene disruptions Aspergillus oryzae acetamidase (amdS) gene recovers pyrG auxotrophs.
Culture medium
YPM culture mediums are by the yeast extract of 10g, the bactopeptone of 20g, the maltose of 20g and complement to The deionized water of 1000ml is constituted.
LB plates be by the bacto-tryptone of 10g, the yeast extract of 5g, the sodium chloride of 10g, 15g bacterium fine jade Fat and complement to 1000ml deionized water constitute.
LB culture mediums and are complemented to by the sodium chloride of the bacto-tryptone of 1g, the yeast extract of 5g and 10g The deionized water of 1000ml is constituted.
COVE sucrose plates by the sucrose of 342g, the agar powder of 20g, the COVE salting liquids of 20ml and complement to 1 liter go from Sub- water is constituted.Culture medium is carried out into sterilizing 15 minutes under 15psi by autoclaving.By the culture medium be cooled to 60 DEG C and Addition 10mM acetamides, 15mM CsCl, Triton X-100 (50 μ l/500ml).
For separate COVE-2 plate tubes：30g/L sucrose, 20mL/L COVE salting liquids, 10mM acetamides, 30g/L promises Boolean (noble) agar (Difco, catalog number (Cat.No.) 214220).
COVE salting liquids by 26g MgSO47H2O, 26g KCl, 26g KH2PO4,50ml COVE trace metals Solution and complement to 1000ml deionized water constitute.
COVE trace metal solutions by 0.04g Na2B4O7·10H2The CuSO of O, 0.4g4·5H2The FeSO of O, 1.2g4· 7H2The MnSO of O, 0.7g4·H2The Na of O, 0.8g2MoO4·2H2The ZnSO of O, 10g4·7H2O and supply the deionization of 1000ml Water is constituted.
Example 1：Differentiate and clone carrys out the lipase gene that autoflash must be mould
Carry out autoflash by QIAamp DNA blood mini kit (Kai Jie companies, Xi Erdeng, Germany) separation mould (to come From Britain, the bacterial strain CBS 304.58,1958 of separation) chromosomal DNA.The chromosomal DNA of 5ug is delivered into FASTERIS SA, Switzerland is sequenced.ORFs for coding lipolytic enzyme analyzes genome sequence, and identifies that flash of light must mould fat Fat enzyme gene D234Z5 (SEQ ID NO:1).From the DNA sequences encoding (CDS) of D234Z5, translated protein sequence (SEQ ID NO:2).Based on SEQ ID NO:2 protein sequence, must mould fat using this flash of light of design coding according to aspergillus oryzae codon CDS (the SEQ ID NO of the synthesis of fat enzyme:3), and it is put into pUC57 carriers.In order to be subcloned into expression vector, by this Synthetic gene is expanded with the primer shown in table 1.
*Capitalization indicates 5'- the and 3'- regions of gene to be amplified, and lowercase is in pCaHj505 carriers Insertion position
It is homologous with carrier sequence at point.
For PCR amplifications, the primer pair (each in forward and reverse) of 20pmol, the PCR are used in PCR reactions Reaction is made up of the following：1 μ l including SEQ ID NO:3 DNA, the 5X GC buffer solutions of 10uL, 1.5uL In dATP, dTTP, dGTP and dCTP of DMSO, 2.5mM each, and 0.6 PhusionTM high fidelity DNA of unit gathers Synthase, final volume is 50 μ l.Amplification is to use Peltier thermal cycler (MJ researchs Co., Ltd, South San Francisco, Jia Lifuni Sub- state, the U.S.) carry out, it is programmed for：It is denatured 1 minute at 98 DEG C；10 circulation, at 98 DEG C be denatured 15 seconds, at 65 DEG C Annealing 30 seconds (wherein each circulation reduces by 1 DEG C) and extension 90 seconds at 72 DEG C；And 26 other circulations, each comfortable 98 DEG C Lower 15 seconds, at 60 DEG C 90 seconds at 30 seconds and 72 DEG C；It is final at 72 DEG C to extend 10 minutes.Then heat block is gone into 4 DEG C of immersions Circulation.
PCR primer is separated by 0.7% agarose gel electrophoresis using tbe buffer liquid, wherein seeing 1.1kb under w light Product band.Then, by using GFX PCR DNA and Gel Band Purification Kit (Gel Band Purification Kit) (GE Medical Groups (GE Healthcare), Buckinghamshire, Britain) is purified into these PCR primers from solution.
With from NEB's (New England's biology laboratory (New England Biolabs), Frankfort, Germany) BamHI and XhoI digested plasmids pCaHj505 (WO 2013029496), and it is solidifying by 0.7% agarose using tbe buffer liquid Gel electrophoresis separate the fragment for producing, and use GFX PCR DNA and Gel Band Purification Kit (GE Medical Groups (GE Healthcare), Buckinghamshire, Britain) purified.Cloning Kit (clone technology is dehydrated by with IN-FUSIONTMCF Laboratories, Inc (Clontech Laboratories, Inc.), mountain scene city, California, the U.S.) connection, will The PCR primer of this purifying of 60ng is cloned into the expression vector pCaHj505 of digestion before of 200ng.
The connection mixture of the dilution of 2.5uL volumes is used to convert Escherichia coli TOP10 Competent cells.From every LB agarose plates of the ml comprising 100 μ g ampicillins selects 4 bacterium colonies, and is confirmed with vector primer by bacterium colony PCR.It is logical DNA sequencing vector primer is crossed (by Nuo Sai genes Co., Ltd (the SinoGenoMax Company of BeiJing, China Limited), Beijing, China) checking flash of light must mould lipase synthesis sequence.Selection be designated as D235M8#1 including SEQ ID NO:3 plasmid is used to be carried out in Aspergillus oryzae host cell MT3568 the different of the lipase of protoplast transformation and its coding Express in source.Recombination bacillus coli transformed cells are trained in 3ml, every ml is supplemented with the LB culture mediums of the ampicillin of 100 μ g Support overnight.It is (triumphant mini preparative (Spin Miniprep) kit (catalogue 27106) to be rotated using Kai Jie companies (Qiagen) Outstanding limited company (QIAGEN GmbH), Xi Erdeng, Germany) plasmid DNA purification.
It was found that the mould lipase maturation protein of flash of light palpus is by 340 amino acid (SEQ ID NO:2 amino acid/11 is to 340) group Into.It is 6.8 to calculate isoelectric point.
Example 2：The genetic transformation Aspergillus oryzae host cell of lipase that must be mould come autoflash with coding
According to WO 95/002043, the protoplast of aspergillus oryzae MT3568 is prepared.By the protoplast and 2.5-10 of 100 μ l The aspergillus expression vector including D235M8#1 (example 1) of μ g and 60%PEG 4000, the 10mM CaCl of 250uL2And 10mM Tris-HCl (pH 7.5) mix, and are gently mixed.Mixture is incubated 30 minutes and these are former at 37 DEG C Raw plastid is applied on COVE sucrose plates to be used to select.After being incubated 4-7 days at 37 DEG C, by four spore inoculatings of transformant To in the YPM culture mediums of 3ml.3 days are cultivated at 30 DEG C afterwards, is used4%-20%Tris- glycine gels (English Outstanding company, Carlsbad, California, the U.S. (Invitrogen Corporation, Carlsbad, CA, USA)) pass through SDS- PAGE analyzes nutrient solution, mould must produce the transformant of the maximum amount of recombinant lipase from flash of light to identify.
Use olive oil/agarose plate (1% albumen quality and grade agarose；1% olive oil；0.008% BG；50mM Hepes；PH 7.2) lipolysis activity of lipase that is produced by aspergillus transformant of investigation.By from different transformant The nutrient solution of 20 μ l aliquots, buffer solution (negative control) are assigned in the punching of a diameter of 3mm and are incubated 1 at 37 DEG C Hour.It is then checked for around the hole in these flat boards presence or absence of the dark green area corresponding to lipolysis activity.
Based on the two selection standards, the spore of best transformant is coated into the COVE-2 plates for again separate, To separate single bacterium colony.Then single bacterium colony is coated on COVE-2 pipes and is incubated until sporogenesis.
Example 3：The fermentation of the host cell of conversion and must mould production lipase from flash of light
During 3 days under 80rpm stirrings at a temperature of 30 DEG C, by the spore from best transformant in shaking flask Cultivated in the YPM culture mediums of 2400ml.Culture broth is harvested by using 0.2 μm of filter filtering.The zymotic fluid of filtering is used Characterized in enzyme.
First, by (NH4)2SO4Precipitation nutrient solution, is then dialysed under pH 5.5 with 20mM NaAC.Then should Sample is applied to the Q balanced with the NaAC of pH 5.5On fast stream (General Electric's Medical Group).With 16 Times column volume, NaCl concentration gradient is applied from 0 to 0.35M NaCl, is then applied with 5 times of column volumes, until 1M NaCl. Collect and concentrate the part with lipase active.
Example 4：The identification of lipase
According to following methods, lipase of the invention purified in above example is characterized.All reactions all Two parts of formula is carried out.
SDS-PAGE：Using SDS-PAGE, the molecular weight for about 26kDa of lipase is determined.
PNP-C8 is determined：Quantify lipolysis activity using pNP-C8 measure.By the 4- nitrobenzophenones from Sigma Caprylate (C8:21742) as stoste in being dissolved in isopropanol using the final concentration of 16.5mM, then with the buffer solution of required pH or Water dilutes 10 times, and the buffer solution or water include 0.4%Triton X-100,10mM CaCl2。
Mix by using the 20 μ l enzyme samples in 0.5mg/ml or as the water of blank and the substrate working solution of 150 μ l Start the reaction, hereafter read OD405.
PH curves：By 20uL enzyme samples and B＆R buffer solutions (Bloomsbury is smooth-Robinson, Robert (Britton-Robinson) buffering Liquid：100mM succinic acid, HEPES, CHES, CAPSO, 1mM CaCl2, 150mM KCl, 0.01%Triton X-100, use HCl With NaOH by pH adjust to 3.0,4.0,5.0,6.0 7.0,8.0,9.0 and 10.0) in 150 μ l pNP-C8 in microtitration Mix in plate and be placed in before the reaction on ice.15 DEG C of Ai Bende constant temperature blendings are set to by the way that microtiter plate is transferred to Instrument starts the measure, and is incubated 40 minutes, hereafter reads OD405.
The optimal pH of lipase of the invention is about pH 8.
Temperature curve
By 20 μ l enzyme samples and pH 7.0, the 150 μ l pNP-C8 in Tris-HCl are mixed in microtiter plate Close, be placed on ice.15 DEG C, 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C are set to by the way that microtiter plate is transferred to Start the measure 40 minutes with 75 DEG C of Ai Bende constant temperature blending instruments of temperature of the measurement, and read OD405.
The lipase has about 30 DEG C of optimum temperature, but also shows good in the scope from 30 DEG C to 75 DEG C Activity.
Heat endurance
Enzyme sample is incubated 0,10,30,60 and 120 minutes and be placed on ice at 50 DEG C.20 μ l enzymes are added to pH 8.0, in the 150 μ pNP-C8 solution with Tris-HCl, and at 150 DEG C be incubated 40 minutes, hereafter read OD405.
At 50 DEG C, the lipase is highly stable.After the testing time most long of 120 minutes is incubated, only slightly Activity reduction.
Example 5：Relative scourability
Automatic mechanical stress measurement (AMSA)：In order to assess the scourability in clothes washing, should using automation Power determines (AMSA) and carries out washing experiment.AMSA flat boards have many seams and lid for test solution, and lid is for all Seam opening strength extruding washing sample (needing the textile for being washed).During wash time, by flat board, test solution, weaving Product and lid high vibration are so that test solution is contacted with textile and applies machinery pressure with rule, periodic swinging mode Power.On further describing, referring to " ad hoc approach embodiment (the Special of WO 02/42740, especially the 23-24 pages Method embodiments) " paragraph.
Enter in the glycine buffer of different pH and in the standard detergent with different surfaces active levels Row clothes washing is tested.Experiment condition is identified below：
Last regulation to specified pH is carried out with NaOH or citric acid.By by CaCl2And MgCl2(Ca2+:Mg2+=4:1) add It is added in test system, the water hardness is adjusted to 15 ° of dH.
After washing, textile is rinsed and excessive water is removed from textile using filter paper in running water, And textile is dried into 5min at 85 DEG C immediately afterwards.
The color change of the dirty textile that scourability is measured as being washed.The spot is the cream mixed with turmeric. Turmeric includes colouring agent curcumin, and it works as pH indicator by with pH dependence color changes.Lipase active Cause that free fatty discharges from butterfat acyl glyceride and this causes pH reductions and thus causes curcumin pH indicator Color change.Therefore lipase scourability can be represented as when being illuminated with white light, anti-from the dirty textile for being washed The discoloration of the light penetrated-launch.
Using professional flatbed scanner (EPSON EXPRESSION 10000XL, Aunar Asia company (Atea A/S), The Barre Shandongs of Lautrupvang 6,2750 are general, Denmark) color measuring is carried out, the scanner is used to capture the dirty textile for being washed Image.In order to extract light intensity value from the image of scanning, by 24 pixel values from image be converted into red, green and Blue (RGB) value.
The color change for being attributed to lipase active is measured as relative to the blueness (B) for reflecting-launching and red (R) light Summation, the increase of the reflection-transmitting of green light (G).Relative to reference lipase (LipolaseTM), the washing performance of lipase Energy (RP (washing)) is calculated as：RP (washing)=(G/ (B+R) (lipase of test)-G/ (B+R) (without enzyme))/(G/ (B+R) (reference lipase)-G/ (B+R) (without enzyme)).
Example 6：Td is determined by differential scanning calorimetry.
Using a VP- capillaries differential scanning calorimeter (micro hot company (MicroCal Inc.), Piscataway, New Jersey, the U.S.) heat endurance that (DSC) determines lipase of the present invention is determined by Differential Scanning Calorimetry.At 200K/ hours Constant programmed heating rate under, heat the heat obtained after enzyme solutions in buffer solution (50mM HEPES, pH 8.0) In analysis chart (Cp is to T), by thermal denaturation temperature Td (DEG C) as the top for being denatured peak (main endothermic peak).
Sample solution and reference solution (about 0.2mL) are continued from 10 DEG C to be filled under 3 hours conditions of storage of (mixing) It is downloaded to (reference solution in calorimeter：Buffer solution without enzyme), and hot pre-equilibration about 20 minutes at 20 DEG C, then from 20 DEG C DSC scannings are carried out to 100 DEG C.Denaturation temperature as 49 DEG C is determined with about +/- 1 DEG C of accuracy.

Claims (17)

1. a kind of polypeptide of the separation with lipase active, the polypeptide of the separation is selected from the group, and the group is made up of the following：
A kind of (a) polypeptide, the polypeptide and SEQ ID NO:2 mature polypeptide have at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%th, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity；
A kind of (b) polypeptide by following polynucleotide encoding, the polynucleotides under low stringency condition, under middle stringent condition, in- With (i) SEQ ID NO under high stringency conditions, under high stringency conditions or very under high stringency conditions:1 or SEQ ID NO:3 or (i) Total length complement hybridization；
A kind of (c) polypeptide, the polypeptide by with SEQ ID NO:1 or SEQ ID NO:3 mature polypeptide encoded sequence has at least 60%th, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence one The polynucleotide encoding of cause property；
D a kind of () polypeptide, the polypeptide is to include substitution, missing at one or more (for example, a number) position, and/or insert SEQ ID NO:2 variant；And
A kind of (e) polypeptide, the polypeptide be (a), (b), (c) or (d) polypeptide of any one of fragment.
2. polypeptide according to claim 1, the polypeptide is not changed in or is included in SEQ IDNO:One or many of 2 Substitution at individual (for example, several) position.
3. the polypeptide according to any one of claim 1-2, wherein the number for replacing be 1-50,1-40,1-30,1-20, 1-10,1-5, such as 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25, 26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、
44th, 45,46,47,48,49 or 50 substitutions.
4. the polypeptide according to any one of profit requires 1-2, the wherein mature polypeptide by with SEQ IDNO:2 amino acid/11 is extremely 340 corresponding residue compositions.
5. a kind of composition, said composition includes the polypeptide according to any one of claim 1-4.
6. composition according to claim 5, said composition further includes at least one surfactant, at least one Surfactant system, at least one soap, or its any mixture.
7. the composition according to any one of claim 5 or 6, wherein these surfactants or surfactant system Selected from anion surfactant, cationic surfactant, nonionic surfactant, amphoteric surfactant, it is facultative from Sub- surfactant, semi-polar nonionic surfactants or its any mixture.
8. the composition according to any one of claim 5-7, wherein said composition are clear laundry cleaning composition, tableware Cleansing composition, hard-surface cleaning compositions and/or personal care cleansing compositions.
9. the composition according to any one of claim 5-8, wherein said composition to be formulated as a kind of conventional, compression Or concentration liquid；A kind of gel；A kind of cream；A kind of soap bar；A kind of conventional or compression powder；A kind of granular solids； Uniform or multilayer tablet of the one kind with two or more layers (identical or different phase)；It is a kind of with one or more rooms Bag；A kind of single or multiple room unit dosage forms；Or its any combinations.
10. a kind of method for hydrolyze lipid, including make the lipid with the polypeptide according to any one of claim 1-4 Or the composition contact according to any one of claim 5-9.
11. is a kind of for cleaning the method for tested material, including makes to be present in lipid spot and the root having in tested material to be cleaned Contacted according to the polypeptide any one of claim 1-4 or the composition according to any one of claim 5-9.
A kind of 12. polynucleotides of the separation for encoding the polypeptide according to any one of claim 1-4.
13. polynucleotides as claimed in claim 12, wherein modifying the polynucleotides with corresponding to the restructuring being intended to for enzyme The codon of the host cell of production is used.
A kind of 14. nucleic acid constructs of the polynucleotides including according to any one of claim 12 or 13.
A kind of 15. expression vectors, the expression vector includes the multinuclear of polypeptide of the coding according to any one of claim 1-4 Thuja acid.
A kind of 16. host cells, the host cell includes the multinuclear of polypeptide of the coding according to any one of claim 1-4 Thuja acid.
A kind of 17. methods for producing the polypeptide according to any one of claim 1-4, the method includes：(a) be suitable to it is many Host cell as claimed in claim 16 is cultivated under conditions of peptide expression；And (b) reclaims the polypeptide.
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